# Dual picosecond fast tunable optical parametric amplifier laser system for wide-field nonlinear optical microscopy

Federico Vernuccio, Assia Benachir, Eric Michele Fantuzzi, Benoit Morel, Simone Bux, Erwann Martin, José Villanueva, Yoann Pertot, Nicolas Thiré, Sandro Heuke, et al.

► To cite this version:

Federico Vernuccio, Assia Benachir, Eric Michele Fantuzzi, Benoit Morel, Simone Bux, et al. Dual picosecond fast tunable optical parametric amplifier laser system for wide-field nonlinear optical microscopy. APL Photonics, 2024, 9 (9), 10.1063/5.0221283. [https://doi.org/10.1063/5.0221283](https://doi.org/10.1063/5.0221283)

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# Dual Picosecond Fast Tunable Optical Parametric Amplifier Laser System for Wide-field Nonlinear Optical Microscopy

Federico Vernuccio,<fnref n="1" /> Assia Benachir,<fnref n="1" /> Eric Michele Fantuzzi,<fnref n="1" /> Benoit Morel,<fnref n="2" /> Simone Bux,<fnref n="2" /> Erwann Martin,<fnref n="2" /> José Villanueva,<fnref n="2" /> Yoann Pertot,<fnref n="2" /> Nicolas Thiré,<fnref n="2" /> Sandro Heuke,<fnref n="1" />,* Hervé Rigneault<fnref n="1" />,*

Authors to whom correspondence should be addressed: sandro.heuke@fresnel.fr; herve.rigneault@fresnel.fr

**ABSTRACT:** In the last decades, nonlinear optical microscopy techniques, such as two-Photon Excited Fluorescence (2PEF), Sum Frequency Generation (SFG), and Hyperspectral Coherent Anti-Stokes Raman Scattering (H-CARS) microscopy, emerged as powerful tools to image biological samples in a label-free, fast, and non-destructive way. Traditionally, these techniques are performed with single point-scanning acquisition schemes featuring a limited field of view and often leading to sample damage when high laser power is used to increase the acquisition speed. Wide-field illumination and camera-based detection schemes offer a solution to these issues enabling fast imaging over a large field view. However, current laser sources used for nonlinear microscopy operating at several MHz repetition rates are not able to induce nonlinear effects over areas larger than 100x100 µm<fnref n="2" />. Here, we present a powerful 200 kHz repetition rate laser source based on an Ytterbium fiber laser pumping two picosecond optical parametric amplifiers (OPA) tunable in the 700-900 nm spectral range, and rapidly tunable (up to 100 KHz) within a 20 nm sub-range. We exemplify the possibilities of this laser system to perform rapid CARS spectroscopy (2 ms/spectrum) and nonlinear wide-field imaging, up to 3.3 frames/s for 2PEF and SFG, and 0.3 hypercubes/s for H-CARS, over a field of view >300x300 µm<fnref n="2" />.

## I. INTRODUCTION

Recent years have seen nonlinear optical microscopy (NLOM) as a powerful tool to visualize different endogenous and exogenous structures in biological samples. Nonlinear optical effects are distinguished into non-parametric and parametric <fnref n="1" />,<fnref n="2" />. In non-parametric processes, the initial and final quantum states of the active molecules are different. This is the case for multiphoton fluorescence imaging techniques that visualize exogenous fluorophores or autofluorescence endogenous molecules in cells and tissues. Among these techniques, the most common one is two-photon excited fluorescence (2PEF), which was demonstrated for the first time in 1990 using a dye laser ³. The same concept of 2PEF also applies to three-photon excitation fluorescence (3PEF) that enables reaching an even longer penetration depth in the samples than 2PEF thanks to the longer wavelengths employed for its excitation ⁴. On the other hand, parametric processes do not feature any energy exchange between the laser beam and the medium, hence, the initial and final quantum states of the active molecules remain the same. Harmonic generation effects belong to this class of nonlinear processes and are mainly used to visualize structural details in the sample. Second-harmonic generation (SHG), first demonstrated in 1961 ⁵, visualizes non-centrosymmetric molecular structures in tissues, such as collagen and muscle fibers. Third-harmonic generation processes (THG) occur in samples

<footnote n="1">¹ Aix Marseille University, CNRS, Centrale Med, Institut Fresnel, Marseille, France.</footnote>

<footnote n="2">² Fastlite by Amplitude, 165 rue des cistes, 06600 Antibes, France.</footnote>
where there is a change in the refractive index or third-order susceptibility in the sample, such as at the water/lipid interfaces of tissues. THG imaging was demonstrated in 1997 6 and is mainly used to visualize lipids, myelin, and cell membranes, in addition to bones and other mineralized tissue interfaces.

Coherent anti-Stokes Raman scattering (CARS) is another parametric process, suitable to visualize the chemical information in biological tissues 7,8. In 1982, M. D. Duncan et al. reported the first work of CARS imaging using gas lasers 9, while in 1999 A. Zumbusch et al. realized CARS imaging using solid-state lasers 10. Contrarily to 2PEF, 3PEF, and SHG, where a single laser beam is enough to generate the nonlinear effect, CARS, in its simplest implementation, exploits two picosecond laser beams, namely the pump ($\omega_p$) and the Stokes ($\omega_s$), that are spatially and temporally synchronized at the sample plane such that the difference in their optical frequencies matches the vibrational resonance $\Omega = \omega_p - \omega_s$ of the sample. In this configuration, a single vibrational mode at a time may be probed, which is not sufficient to distinguish different molecular species in heterogeneous biological samples. This calls for hyperspectral CARS (H-CARS) approaches where one of the two laser sources is rapidly tuned to sequentially acquire multiple vibrational modes 11.

Most of these NLOM techniques have been implemented in a point-scanning fashion, employing a scanning system either based on galvanometric mirrors or a motorized translational stage, and large area single-element detectors - commonly multi-alkali photomultiplier tubes (PMTs) or avalanche photodiodes (APDs). However, point-scanning schemes are limited in speed and sample damage may occur when high laser power is used to increase the acquisition speed. Wide-field illumination schemes, achieved using powerful laser sources and camera-based signal collection, simplify the setup complexity and enable fast imaging over a large field of view. However, the available peak power of the employed laser sources for a given exposure time limits the field of view of these imaging techniques to $\approx 100 \times 100 \ \mu m^2$ 12–18. In these implementations, one could increase the field of view, reducing the power density at the sample, and thus the nonlinear signal, and compensate by increasing the measurement time. However, if the exposure time is long enough, the noise from the dark current may also start to become a larger portion of the signal.

To overcome the current limitations in wide-field multiphoton and coherent Raman imaging, the employed laser system needs to satisfy the following requirements: (i) disposing of at least two beams, one of which is tunable to probe different vibrational modes of the scrutinized sample; (ii) having enough pulse energies (tens of microjoules) to induce nonlinear effects at the sample plane, hence, a low operating repetition rate of the laser system (100 KHz - 1 MHz) is preferred to a high one (20 MHz - 80 MHz) conventionally used in point-scanning schemes; (iii) having a pulse duration of $\approx 1-2$ picosecond to spectrally resolve the different vibrational modes at the sample plane (10-20 cm$^{-1}$ in condensed media).

The advances in NLOM have been closely linked to the development of ultra-fast lasers delivering pulse trains with sufficiently high peak power to activate the nonlinear processes. The first demonstration of mode-locked titanium-doped Sapphire lasers in 1992 19 was crucial for NLOM. Since its invention, other solid-state laser media, such as Nd:glass 20, Yb:glass 21, Yb:tungstate 22, and Cr:forsterite 23, have also been developed. These sources have enabled the dramatic growth of multiphoton and harmonic-generation imaging 24–26. Standard Ti:sapphire oscillators are tunable in the range 700–1000 nm, with pulse energies in the range 20–30 nJ, pulse durations under 100 fs, repetition rates around 100 MHz, and average powers of 2 W. Moreover, solid-state lasers can be coupled with optical parametric oscillators (OPOs) to further extend the operating wavelength region for THG and 3PEF imaging 27, as demonstrated for the first time in 1993 by T. F. Albrecht et al. 28.
Fiber laser systems have also become particularly suitable for NLOM starting from the late
90s with applications in multi-harmonic ²⁹ and multiphoton fluorescence imaging. Fiber lasers
avoid precise optical alignment, enable lower repetition rates than Ti:sapphire lasers thanks to
their ability to readily enlarge the fiber cavity length without increasing the overall footprint,
and can provide high power ³⁰. Despite all these advantages, short-pulse fiber lasers do not
guarantee a range of tunability as large as solid-state lasers, essentially limited by the availability
of the fiber gain media. The tunability limitation can be overcome when they are used in
combination with free-space optical parametric amplifiers (OPAs) and OPOs ³¹. Though fiber-
based (F)OPAs and (F)OPOs exist ³²,³³, the handling of high peak powers often creates
undesired side effects (e.g., stimulated Raman gain and self-phase modulation) that modify the
spectral and temporal pulse shape. OPAs operate with lower repetition rate (typically from 1 to
100 kHz) systems, requiring high pump intensities. On the other hand, small-scale femtosecond
oscillators can pump OPOs and may provide pulses with high repetition rates (100 MHz) ³⁴,³⁵,
benefiting experiments where weak signals need to be detected through modulation transfer
techniques ³⁶ (as in pump-probe schemes). However, OPAs are quite easy to operate and provide
higher energy pulses, and broader and faster tunability than OPOs, since no cavity length
stabilization is required.

Until recently most of the systems for NLOM used laser systems at high repetition rates (10-100 MHz) and OPOs with pulse energies in the nanojoule range. These lasers do not fulfill the requirements for wide-field nonlinear microscopy. The demonstration of 3PEF for neuroscience applications has pushed the development of low repetition rate systems (100 kHz – 2 MHz) with µJ power to perform deep tissue imaging ²⁷. These systems are based on high-power (10-100 W) Ytterbium laser pumping OPAs that can provide several Watts of power at hundreds of kHz repetition rate in the 700nm – 1.7µm range and sub-70 fs pulse duration ³⁷. Recently, we demonstrated that an amplified fs-fiber laser system in combination with an OPA at 500kHz can perform multiphoton and CARS wide-field imaging ¹². Fs-laser systems are suitable for inducing multiphoton processes, such as TPEF and multi-harmonic generation, over a large field of view. Moreover, they can be used to perform wide-field CARS imaging but do not provide sufficient spectral resolution to distinguish the different vibrational bands in heterogeneous samples, unless more sophisticated detection schemes (e.g., based on Fourier transform techniques ³⁸) are used in detection. Ps-systems are more suitable for guaranteeing sufficiently good spectral resolution in CARS, reducing sample phototoxicity, and simplifying the detection scheme. However, to maintain the same signal extraction of fs-laser systems over a large field of view for a given repetition rate, ps systems must have a higher average power to induce nonlinear effects. Nowadays, high-peak and average power ps-laser systems with fast tunability are commercially unavailable, thus preventing wide-field NLOM with high spectral resolution and large field of views.

To break through this limitation, we present here the development and characterization of a
dual-OPA laser system demonstrating its suitability and versatility for wide-field nonlinear
optical microscopy. Our system delivers two independent picosecond laser pulses tunable from
700 to 900 nm and rapidly tunable (up to 100 kHz color-sweeping rate) within a 20-nm sub-
range with pulse energies >15 µJ (a factor of 1000 higher than the pulse energies employed for
point scanning microscopy) at 200 kHz repetition rate. We test the OPA sources, combining
them with picosecond Stokes pulses at 1030 nm, to perform CARS spectroscopy (2ms/spectrum
over 300cm⁻¹), wide-field 2PEF, sum frequency generation (SFG) (up to 3.3 frames/s) and wide-
field CARS microscopy (up to 33 frames/s) over a field of view larger than 300x300 µm². This
field of view is 20 times larger than what is reported in the wide field CARS microscopy
literature [12, 13,16]. Eventually, we demonstrate for the first time H-CARS wide-field microscopy over 300x300 µm² in 3.3 s (0.3 hypercubes/s) over a bandwidth of 300cm⁻¹.

## II. METHODS

### II.I SAMPLE PREPARATION

Preparation of the lamb tendon tissue sample: 10 µm thick unstained tissue sections from a lamb tendon were cut and deposited on a 1 mm thick glass coverslip. A droplet of deionized water was added on top of the tissue to reduce the linear scattering of the sample. To avoid draining the water, the tissue sample was sealed using a 100 µm thick spacer and a cover slip.

Preparation of U2OS cells with Alexa Fluor 568: U2OS osteosarcoma cells were maintained in McCoy's medium (Gibco, 16600082) supplemented with 10% fetal bovine serum (Dominique Dutscher, S181H), 100 U/mL penicillin, and 100 µg/mL streptomycin (Sigma-Aldrich, P4333) in a humidified incubator with 5% CO₂ at 37 °C. Cells were seeded onto a sterilized glass coverslip and allowed to attach and spread for 16 h. Subsequently, cells were fixed for 15 min with 4% paraformaldehyde (Electron Microscopy Sciences, 15714) in cytoskeleton buffer (10 mM MES pH 6.1, 150 mM NaCl, 5 mM EGTA, 5 mM glucose, 5 mM MgCl₂). Fixed cells were incubated with 165 nM Alexa Fluor 568 phalloidin (Thermo Fisher Scientific, A12380) in 0.1% saponin/1% BSA/PBS for 1 hour to stain F-actin. After three 10-minute washes with 0.1% saponin/1% BSA/PBS, followed by two 5-minute washes with PBS, the coverslip with stained cells was mounted with Fluormount aqueous mounting medium (Sigma-Aldrich, F4680) and stored at 4 °C until image acquisition.

Preparation of drosophila larva samples: Larvae were dissected in cold PBS, fixed in 4% PFA for 30 minutes, and rinsed in 1% Triton. E-Cadherin was labeled with antibodies (primary: Rat anti-Ecad, DSHB DCAD2; secondary: anti-rat Alexa-488). Actin was labeled with 1h incubation in 0.4 µM Phalloidin-alexa-568 (Thermofisher). Nuclei were labelled with a 10-minute 1/1000 hoechst incubation.

For the experiments with polystyrene (PS) beads and immersion oil, we adopted a sample sandwich configuration. We deposited small quantities (≈8 µl) of PS beads diluted in water onto a 170 µm thick glass coverslip, waited for them to dry, and then released a ≈10 µl droplet of immersion oil, sealing the second coverslip with enamel.

## III. RESULTS

### III.I LASER SYSTEM

The laser system is based on a powerful Ytterbium (Yb) laser at 1030nm operating at 200 kHz and pumping 2 OPA lines. In the first step, the laser generates a broad supercontinuum in a bulk YAG crystal from which 2 independent fast-tunable acousto-optic filters select 1-1.6 nm bandwidth as seeds for the 2 OPA lines. The seed lasers are amplified in two steps via optical parametric amplification pumped by a frequency-doubled laser beam at 515nm.

More precisely, the Ytterbium (Yb) fiber laser (Tangor 100, Amplitude) delivers ≈450 fs pulses at 1030 nm and 100 W average power. The repetition rate (rep rate) of this Yb laser can be tuned from single shot up to 40MHz. For the results presented in this work, the rep rate is set at 200 kHz. In the supplementary information, we present the optical scheme and the characterization measurements of the laser sources when setting the repetition rate at 1 MHz (See Figure S2 and S3). The Yb laser output is negatively chirped to reach ≈1.5 ps pulses and is used to pump two Optical Parametric Amplifier (OPA) lines (named hereafter ‘line 1’ and ‘line 2’), both operating simultaneously at the rep rate of the Yb laser (200kHz – 500 µJ or 1MHz-100 µJ). Figure 1a gives a general overview of the laser. The laser system provides 7 output beams (Fig. 1a) whose individual power can be controlled by rotating a half-waveplate
combined with a fixed polarizing beam splitter. The first output (output 1 in Figure 1a) provides 5W average power at 1030 nm and is generated by propagating the beam into a 95:5 beam splitter, taking the transmitted 5%. The remaining 95% of the 1030 nm power feeds two identical OPA lines (line 1 and line 2) that have one or two amplification stages depending if the rep rate is 1MHz or 200kHz, respectively.

In the 200 kHz rep rate configuration (shown in Figure 1a), a 80:20 beam splitter is added to reflect 80% of the laser power towards a 2 mm thick barium borate (BBO, 10x10 mm² aperture, theta=23.4°) crystal (named ‘SHG stage 2’ in Figure 1a) for generating the 515 nm pump beams for the ‘second stages’ of amplification of both OPA lines. The remaining 20% of 1030 nm Yb laser light is further divided through a 90:10 beam splitter. 90% of the optical power is used to generate the 515 nm pumps for the ‘first stages’ of amplification of the two OPA lines in another 2 mm thick BBO (10x10 mm² aperture, theta=23.4°) crystal (named ‘SHG stage 1’ in Fig. 1a), whereas the other 10% is used to generate a supercontinuum into a 10 mm YAG crystal (named ‘Supercontinuum generation’ on Fig. 1a). Both BBO crystals are maintained at constant temperature (60°C) in ovens and have a conversion efficiency of ≈55% for each crystal. Output 2 (Fig. 1a) delivers the depleted 1030 nm beam after the second SHG stage with ≈4 ps pulse duration and 32 W output power. This beam comes from the light transmitted by a dichroic mirror, reflecting the 2ω (515 nm), placed after the ‘SHG stage 2’ (Fig. 1a). Similarly, output 3 (Fig. 1a) delivers the depleted 1030 nm after the first SHG stage with ≈1.5 ps pulse duration and 7 W output power.

<figure><img src="image_2.png" /><figcaption>Figure 1: (a) Laser system in the 200 kHz repetition rate configuration. (b) Normalized spectrum of the blue-shifted spectral part of the supercontinuum (blue curve) and power dependence of the OPA line 1 (Output 5) beam (red curves). (c) Autocorrelation measurements</figcaption></figure>
of Yb 1030nm (Output 1), and OPA line 2 (Output 7) at 700 nm and 900 nm. Legend: BS: beam splitter; DM: dichroic mirror; AOPDF: Acousto-Optical Programmable dispersive filter. (d) Spectral profile of the OPA (Output 5) for different operating wavelengths (700-800-900 nm).

The ‘supercontinuum generation’ unit (Fig. 1a) is constituted of a motorized waveplate and a polarizing beam splitter to finely tune the 1030 nm Yb laser power, while a 125 mm lens focuses this beam onto the 10mm thick YAG crystal placed on a manual translational stage to adjust the focus position along the z-direction. Furthermore, the YAG crystal is mounted on a motorized stage that continuously translates the lateral position of the focused beam within the crystal to avoid damage with a speed of 0.2Hz. The generated broadband supercontinuum (SC) light is filtered to select its blue-shifted spectral part spanning from 600 to 950 nm (see the blue spectrum in Figure 1b). A 50:50 splitter divides the SC into two arms to produce the two seed beams for the first stages of amplification of the two OPA lines. Two acousto-optic programmable dispersive filters (AOPDF, dazzlers, Fastlite) ³⁹ are used to independently select the seed wavelengths of lines 1 and 2 with a full-width half-maximum (FWHM) of around 1-1.6 nm in the range between 700 and 900 nm (Figure 1d). The OPA can operate from 1Hz to 100 kHz in a 20 nm bandwidth, in steps of 0.2 nm, by simply tuning the radiofrequency driving the AOPDF. This 20 nm bandwidth limit corresponds to the OPA lines amplification gain bandwidth that does not require any mechanical nor thermal tuning of the OPA lines.

The 515 nm pump generated at ‘SHG stage 1’ (Fig. 1a) is split into two equal beams by a 50:50 beam splitter. Then, the two 515 nm ‘SHG stage 1’ beams are independently combined with the seed beams in two 10 mm long LBO crystals (theta=90°, phi=9.5°, AR 515-2000 nm, R<1% 515 nm, R<5.5% @2000 nm) to constitute the first OPA stages (OPA1) of line 1 and line 2. OPA1 for both line 1 and 2 operates in a collinear configuration. The idler beams (line 1 and 2) produced in OPA1 are sent using two dichroic mirrors to output 4 and output 6. These outputs are tunable in the range between 1200 nm and 2000 nm with an average power of 1.2 W.

Similarly to what was explained for OPA1, the 515 nm generated at ‘SHG stage 2’ is split with a 50:50 beam splitter to produce the two pump beams for the second OPA stage (OPA2) of line 1 and line 2. OPA2 operates in a non-collinear configuration through a 2-mm thick BBO crystal (10x8 mm² aperture, theta=23°). The signals deriving from the two lines after the two stages of amplification (OPA1 and OPA2) are delivered to output 5 and output 7. These two outputs present similar characteristics regarding pulse duration, FWHM, beam profile, average power at different wavelengths, and power and pointing stability. In particular, we measured an average output power of 5.1 W at 700 nm for both lines and 3.3 W at 900 nm (see spectra in Figure 1b) with pulse duration (pulseCheck, APE) of 715 fs and 878 fs, respectively (see Figure 1c). Power stability measurements carried out over 15 hours with a power meter (PM16-425, Thorlabs) demonstrated an RMS stability of 0.4% for line 1 at 700 nm (Figure 2a). Moreover, Figure 2b reports the far field beam profile measured with a standard CMOS camera (Basler) for OPA line 1 at 700 nm. Further characterization measurements (autocorrelation of the different outputs and pointing stability) of the optical sources at 200 kHz rep rate are reported in the supplementary information (Figure S1-S3).
<figure><img src="image_3.png" /><figcaption>Figure 2 (a) Power stability of OPA line 1 at 700 nm and 200 kHz repetition rate over 15 hours. (b) Far field beam profile of OPA line 1 at 700 nm and 200 kHz repetition rate.</figcaption></figure>

Each OPA line features 5 different parameters that can be tuned to optimize the amplified beam in terms of its spatial profile, average power, and spectral line width. In the first stage of amplification (OPA1), the phase matching within the 10 mm LBO crystal is controlled through a motorized delay line and an oven that adjusts the temperature when varying the wavelength of the seed. In the second stage of amplification (OPA2), phase matching is guaranteed by adjusting the position of a second motorized delay line and the rotation of the BBO crystal through a motorized rotational stage. Finally, average power optimization is achieved by adjusting the amplitude voltage of the RF signal sent to the AOPDF.

The power stability of the OPA lines is mainly related to the stability of the SC white light source which depends on the stability of the pump laser power at 1030nm and the position of the focal plane. The source does not need any daily alignment or variation of the position of the various actuators. The SC stability and the line shape of the amplified seed are monitored through a spectrometer (AvaSpec-Mini4096CL, Avantes). A laser beam stabilization system (MRC) guarantees the pointing stability of the 1030nm Yb laser beam before pumping the two OPA lines and generating the SC. The MCR corrects for slow drifts due to temperature variations in the room. The desired position of the pump laser beam at 1030 nm is defined by a pair of 4-quadrant-diodes (4-QD) that monitor a small portion of laser power transmitted through high-reflective mirrors. The closed-loop controller continuously determines the deviation of the laser beam from the desired position and drives the fast actuators to move the steering mirrors stabilizing the laser beam.

The calibration of the whole laser system consists of saving the optimal set of parameters for the wavelength in the range between 700 nm and 900 nm for each OPA line. We typically save 5 sets of parameters associated with 700, 750, 800, 850, and 900 nm emission wavelengths. Calibration for all the intermediate points is performed by fitting the saved parameters with a polynomial function of the 2^nd^ order.

The OPA line 1 and 2 can be tuned automatically and independently from each other across the 700 - 900 nm spectral range in a few seconds. This broad range spectral tuning speed is limited by the OPA1 oven thermal tuning that requires longer stabilization times for bigger wavelength steps. However, when a set of mechanical positions and temperature of the crystals are selected, rapid tuning can be performed independently at 100kHz over a range of about 20nm using the AOPDF. This enables hyperspectral investigations as explained below.
### III.II SETUP CONFIGURATION FOR CARS SPECTROSCOPY AND MULTIPHOTON WIDE-FIELD IMAGING

We set the system in the 200 kHz rep rate configuration to demonstrate its capability to perform high-speed CARS spectroscopy of test solvents and multiphoton wide-field imaging through 2PEF, harmonic generation (SFG and SHG), and hyperspectral CARS on test samples. For the spectroscopy and wide-field imaging, we used a different configuration for the sample illumination and the signal collection (Figure 3a). We used Output 1 (1030 nm, 1.5 ps) of the laser system as the Stokes beam and either Output 5 (OPA line 1) or Output 7 (OPA line 2) as tunable pump beams in the range 700 to 900 nm. We combined the pump and Stokes pulses using a dichroic mirror (DMLP1000, Thorlabs) and overlapped them in time by moving a mechanical delay stage positioned on the pump path.

For spectroscopy experiments, a 10x objective (Nikon Plan 10x/0.25) is placed after the combiner to focus the pump and Stokes train of pulses on a 1 mm thick cuvette filled with a test solvent. The sample is placed on a mechanical translational stage to adjust the focus. A lens ($f=25.4$ mm) is used for signal collection. After the lens, we placed two short pass filters (FESH0700, Thorlabs) to filter out the Stokes and pump beams and read either in a spectrometer (Flame, Ocean Insight) or a photomultiplier (PMT1001/M, Thorlabs) the generated anti-Stokes CARS signal.

For wide-field microscopy experiments, we reduced the beam spot size of both the pump and Stokes beam with two independent 3:1 telescopes placed before the combiner, thus reaching ≈1 mm spot size at the sample plane. The sample is vertically mounted on an x-y mechanical sample holder and a mechanical translational stage is used to adjust the z-position. The signal is collected through either a combination of a 10× objective (Nikon Plan 10×/0.25) and a 200 mm tube lens, leading to an effective magnification of 10, or a 20× objective (Olympus LUCPlanFL N 20×/0.45) and a 300 mm tube lens, leading to an effective magnification of 33.3. Then, the signal is directed to a CMOS camera (ORCA-Fusion Digital CMOS camera C14440-20UP, Hamamatsu). To avoid generating undesired non-resonant background signals on the collection optical components and to prevent any damage to the CARS-signal-collecting objective lens, we placed a low-pass filter (FESH0700, Thorlabs) between the sample and the objective. Further filters to select the SFG, TPEF, and CARS signal bandwidth (see scheme in Figure 3a) are placed in front of the camera.

### III.III H-CARS SPECTROSCOPY

We assessed the spectroscopic performances of our laser system by measuring the CARS spectrum in the high wavenumber regions (or CH-stretching) (2800 – 3150 cm⁻¹) of test solvents. For all these spectroscopy experiments (figure 3a), we adjusted the laser power of the pump and Stokes beam to have 20 mW and 10 mW, respectively, at the sample plane. We set the center wavelength of the OPA to 790 nm, tuned the seed between 780 and 800 nm (20 nm bandwidth) with the fast AOPDF operating at 10 kHz, and set the number of steps to 100. Therefore, a total spectral span between 780 nm and 800 nm is performed in 0.1 ms * 100 = 10 ms. For the experiments with the spectrometer, we set the exposure time to 30 ms, leaving the spectrometer exposed to acquire the CARS signal of 3 consecutive sweeps of frequencies. For this demonstration, we used dimethyl sulfoxide (DMSO), toluene, and ethanol as test solvents and we compared the measured CARS spectra with spontaneous Raman spectra (SR) (figure 3b). The SR spectra were acquired in 5 seconds with a homemade system using a continuous-wave diode laser (Cobolt AB, Flamenco) centered at 660 nm to drive the Raman signals. Complete details on the SR setup description can be found in our previous works 40,41. The presented CARS spectra correspond to the raw data and no processing has been done to
smooth the spectra or remove the non-resonant background (NRB), derived from the four-wave
mixing processes occurring at the sample. Therefore, the distortions of the line shapes of the
CARS peaks from the SR ones are due to the NRB contribution, which we decided not to
remove as the non-resonant contribution in this part of the vibrational spectrum is negligible as
compared to the resonant one.

We estimated the system response function (SRF) to a Dirac Delta spectral response by
fitting with a Gaussian function the peak of DMSO centered at 2914 cm⁻¹ both for the CARS
and SR spectrum (figure 3b). Assuming that the SR setup resolution (< 4 cm⁻¹) does not perturb
the line shape of the peak, we calculated the SRF as the deconvolution between the CARS
Gaussian fit, featuring a FWHMCARS of 27 cm⁻¹, and the SR one, with FWHMSR= 16 cm⁻¹. The
retrieved spectral resolution of the system is ≈22 cm⁻¹.

To test the fast tunability speed of our laser system, we replaced the spectrometer with a
photomultiplier and measured the DMSO spectrum at various operating frequencies of the
AOPDF (1 kHz, 10 kHz, 20 kHz, and 50 kHz). We synchronized the PMT acquisition with the
RF modulation signal by sending to a fast data acquisition card (vDAQ, MBF Bioscience) the
trigger signal from the RF generator and the signal coming from the PMT. The trigger signal
consists of a train of pulses in which each pulse corresponds to the start of the pump wavelength
sweeping. Since the PMT can resolve the individual laser pulses at 200 kHz, we used a low-
pass electronic filter with 1 kHz bandwidth (EF110, Thorlabs) to remove the high-frequency
components. All these measurements with the PMT have been acquired using ScanImage⁴² in
Matlab 2023b.
<figure><img src="image_4.png" /><figcaption>Figure 3 (a) On the left-hand side, laser sources angular frequencies and the generated nonlinear signals (Stokes: Output 1, pump: Output 5 or 7); on the right-hand side, the experimental scheme for CARS spectroscopy and nonlinear wide-field microscopy. (b) Estimation of the spectral resolution of the system and H-CARS spectra of test solvents acquired with a spectrometer and comparison with spontaneous Raman (SR) data. (c) CARS spectra of DMSO acquired with a PMT sweeping the pump wavelength at different operating frequencies.</figcaption></figure>

Figure 3c showcases the results of these experiments. A higher operating frequency implies a lower integration time to collect the CARS spectrum of DMSO in the whole 2800-3000 cm⁻¹ range. Due to the presence of the electrical low-pass filter, the spectral resolution deteriorates for high AOPDF operating frequencies. Indeed, the number of different spectral colors effectively read by the acquisition card is reduced. This reasoning explains why the peak gets broader and less steep on the falling edge while increasing the operating speed. For each measurement, we calibrated the horizontal axis by considering the linear fit between the positions of the main peaks of DMSO in the CARS and SR spectrum. This justifies why the graphs at 1 kHz and 50 kHz end earlier.
### III.IV NONLINEAR WIDE-FIELD MICROSCOPY

To fully exploit the versatility of our laser system in terms of tunability of the sources and high throughput, we demonstrated nonlinear optical wide-field imaging of test samples using different contrast mechanisms under a plane wave illumination. We set the experiment to the microscopy scheme (figure 3a) and used a 10x magnification imaging system. We fixed the OPA line 2 to 790 nm and the camera exposure time to 300 ms, and acquired SFG images of a 20 µm thick lamb tendon slice deposited on a 1-mm-thick glass coverslip (figure 4a - field of view: 700 µm x 700 µm – 3.3 frames/s). To collect the signal coming from the collagen-rich structures of the scrutinized lamb tendon sample, we placed in front of the camera a low-pass filter (FESH0600, Thorlabs) and a band-pass filter (MF 460/60, Thorlabs) that enabled us to visualize the SFG signal around 447 nm and remove the 515 nm SHG signal generated by the 1030 nm Stokes beam (Output 1) and the 395 nm SHG signal generated by the 790 nm OPA. In this experiment, we used 2 W average power of the Stokes beam and 2 W of the pump beam. These values of average power at the sample plane enabled us to obtain a good image contrast without experiencing any sample damage.

We also tested the setup with 2PEF experiments. We first used the 1030 nm Yb laser beam (Output 1) and 2 W average power to illuminate a sample of U2OS cells treated with Alexa Fluor 568, a fluorescent dye used to visualize F-actin in tissue sections, cell cultures, or cell-free actin preparations. For 2PEF experiments, we increased the detection scheme magnification to 30x, by combining a 20x objective lens (Olympus LUCPlanFL N 20x/0.45) with a 300 mm tube lens. Figure 4b shows the 2PEF image, highlighting the distribution of F-actin in cells over a field of view of 215 x 215 µm² and 1s exposure time.

Next, we prepared a sample of a larval epidermis of drosophila treated with three different dyes: Hoechst which stains the nuclei, Alexa Fluor 488 which stains cadherins, which is essential in maintaining cell-cell contact and in regulating cytoskeletal complexes, and Alexa Fluor 568 which attaches to the protein-rich structures of the sample. After sample treatment, a ≈20 µm thick slice of larva was deposited on a 1 mm coverslip and imaged with the same setup configuration adopted to image the U2OS cells.
<figure><img src="image_5.png" /><figcaption>Figure 4 (a) 700x700 µm² SFG image of lamb tendon highlighting the collagen structures (3.3 frames/s). (b) 300x300 µm² 2PEF image of U2OS cells treated with Alexa Fluor 568 obtained with the 1030 nm Stokes beam (1 frame/s) and 2x zoom to visualize the actin filaments. (c) 300x300 µm² 2PEF images of the larval epidermis of drosophila treated with three different fluorophores (Hoechst, Alexa Fluor 488, and Alexa Fluor 568) excited with three different wavelengths OPA line 2 (Output 7) (700, 750, and 800 nm). The large panel shows the composite image deriving from the three frames that were acquired sequentially. Scale bar: 50 µm. Camera integration time is 300ms for the Alexa dyes and 2s for Hoechst.</figcaption></figure>

We blocked the 1030 nm beam and used three different OPA wavelengths to excite the three different fluorophores. For imaging the abundance of Hoechst in the sample (blue panel in
Figure 4c), we tuned the OPA line 2 to 700 nm with 2 W average power at the sample plane. In front of the camera, we placed a short pass filter (FESH0600, Thorlabs) in combination with a band pass filter centered at 416 nm and 46 nm bandwidth (BP416-46). For imaging the Alexa Fluor 488 (blue panel in figure 4c), we tuned the OPA line 2 to 750 nm with 2 W average power at the sample plane. We replaced the band-pass filter used for imaging Hoechst with a bandpass filter centered at 530 nm and 55 nm bandwidth (BP530-55). Eventually, for imaging the Alexa Fluor 568 (red panel in Figure 4c), we tuned the OPA line 2 to 800 nm with 2 W average power at the sample plane. In front of the camera, we inserted a short-pass filter (FESH0700, Thorlabs) in combination with a bandpass filter centered at 618 nm and 50 nm bandwidth (BP618-50).

These images highlight the different compounds marked by the fluorophores, i.e., nuclei, cadherins, and protein-rich structures, of the muscles underlying the larval epidermis of the drosophila. The three frames were acquired setting the camera exposure time to 300 ms for the case of Alexa Fluor 488 and Alexa Fluor 568 and to 2 s for the Hoechst.

A composite image highlighting the palette contrast of the three different channels is shown on the right-hand side of Figure 4c.

<figure><img src="image_6.png" /><figcaption>Figure 5. Hyperspectral CARS imaging over the entire CH-stretching region of 10 µm polystyrene beads in immersion oil. The plot shows the raw single-pixel spectra of the polystyrene (blue curve) and the immersion oil (orange curve). Scale bar: 50 µm; single frame exposure time: 30 ms (33 frames/s).</figcaption></figure>

### III.V HYPERSPECTRAL CARS WIDE-FIELD MICROSCOPY

Next, we combined the fast AOPDF wavelength tunability over the 20 nm range of the laser system with the provided high pulse energies to perform hyperspectral wide-field CARS images over the entire CH-stretching region. We imaged a sample of 10 µm polystyrene (PS) beads in oil (objective immersion oil) sandwiched between two 170 µm thick glass coverslips. We centered the OPA line 2 wavelength to 790 nm and operated the AOPDF at 30 Hz with 100 steps and 20 nm bandwidth. Therefore, a total spectral span between 780 nm and 800 nm is performed in 33 ms x 100=3.3 s. We set the camera exposure time to 33 ms and acquired a stack of 100 images on the same field of view. We designed the detection system with a 10x magnification
and placed in front of the camera two filters (FESH0700 and FELH0550, Thorlabs) to read only the CARS signal.

Figure 5 showcases the normalized single-pixel CARS spectra associated respectively with polystyrene and immersion oil. Moreover, we report 4 different frames corresponding with 4 different Raman shifts in the high wavenumber regions (2855 cm⁻¹, 2890 cm⁻¹, 2950 cm⁻¹, and 3050 cm⁻¹), also highlighted with colored vertical bars in the spectra plot. The frames are directly derived from the raw data after normalization of the whole dataset over the global maximum and rescaling over the whole 16-bit camera dynamic. The panels at different Raman shifts show a different contrast between the two chemical species (PS beads and oil) varying the excitation wavelengths. This demonstrates the ability of the laser system to provide 0.3 Hz hyperspectral CARS imaging over a field of view larger than 300x300 µm². An example of a video of a real-time hyperspectral CARS experiment is reported as a .mp4 file in the supplementary information.

#### IV. CONCLUSION

We have developed an original dual optical parametric amplification (OPA) system based on a 100 W powerful Yb fiber laser to pump two independent optical parametric oscillator (OPA) lines capable of delivering tunable laser trains of 800 fs pulses with up to 5 W average power and 200 kHz repetition rate in the range between 700 and 900 nm. The system can also operate at a 1 MHz repetition rate and deliver ~2.5 W average power with a 800 fs pulse duration and the same spectral tunability. Not only does the system tune the wavelength in the entire spectral range within a few seconds, but it can also scan the wavelengths within a limited 20 nm bandwidth with operating frequencies from 1 Hz to 100 kHz, thanks to the seed beam extraction from a supercontinuum by fast acousto-optic tunable filters. We have exemplified the laser fast tunability combined with its high pulse energies to perform fast CARS spectroscopy (up to 50 kHz) and 2PEF and SFG wide-field imaging over a field of view of 300x300 µm². We combined the fast spectroscopy and imaging capabilities to perform hyperspectral CARS images in the entire CH stretching region (2800 cm⁻¹-3050 cm⁻¹) at 0.3 Hz. To our knowledge, the reported laser system provides the fastest hyperspectral imaging over such large (300x300 µm²) field of view. The latter is from 20 to 200 times larger than previously reported wide-field CARS implementations [12-16].

The laser system can also operate in the fingerprint region for wavenumbers smaller than 1400 cm⁻¹ using the combination of the two OPAs as pump and Stokes beams, however, NRB removal algorithms are needed to process hyperspectral CARS images and deliver false color chemical maps. We demonstrated the wide-field ability of the developed laser system using a simple plane wave illumination scheme that is sub-optimal in terms of image quality, resolution, and z-sectioning capability compared to conventional point scanning schemes. However, wide-field approaches such as structured illumination⁴³ or random illumination microscopy (RIM)¹² can overcome point scanning in terms of speed, spatial resolution (2-fold improvement), and field of view. The flexibility of the developed laser system will allow for the exploration of these more advanced wide-field nonlinear techniques, particularly beneficial for visualizing morpho-chemical details in biological applications such as coherent Raman histology⁴⁴,⁴⁵.

**FUNDING.** We acknowledge financial support from the Centre National de la Recherche Scientifique (CNRS), A\*Midex (ANR-11-IDEX-0001-02), ANR grants (ANR-10-INSB-04-01, ANR-11-INSB-0006, ANR-16-CONV-0001, ANR-21-ESRS-0002 IDEC), INSERM 22CP139-00. This has received funding from European Union's Horizon 2020 (EU ICT 101016923 CRIMSON), Horizon Europe (Marie
Skłodowska-Curie Postdoctoral Fellowship 101148683 CHIMERA), and European Research Council (ERC, SpeckleCARS, 101052911). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council. Neither the European Union nor the granting authority can be held responsible for them.

**ACKNOWLEDGMENTS.** The authors thank Annafrancesca Rigato for providing the drosophila samples, and Shizue Omi-Massieu for preparing the U2OS cells.

**DISCLOSURES.** The authors declare no conflict of interest.

**AUTHOR CONTRIBUTIONS.** H.R. conceived the project; F.V. designed and built the wide-field nonlinear optical microscope and conducted the spectroscopy and microscopy experiments with the help of A.B., E.M.F., S.H, and H.R.; N.T., B.M., S.B., and Y.P. designed and built the dual-OPA system with the guidance of H.R.; E.M. and J.V. developed the software and the electronics of the laser system with the help of N.T.; F.V., S.H., and H.R. wrote the manuscript, with the inputs of all authors. All authors have approved the final version of the manuscript.

**DATA AVAILABILITY.** The data that support the findings of this study are available from the corresponding author upon reasonable request.

**SUPPLEMENTAL DOCUMENT.** See Supplement 1 for supporting content.

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