Optical imaging, particularly coherent Raman scattering (CRS), offers chemically specific, label-free contrast with high resolution and sensitivity, making it suitable for biomedical applications. CRS microscopy allows for minimally invasive, continuous live imaging of biomolecules, overcoming limitations of fluorescence-based methods such as fluorophore bleaching and phototoxicity. However, clinical translation of CRS has been hampered by the high cost, bulkiness, and complexity of conventional laser sources like Ti:sapphire lasers and OPOs. These typically require vibration-isolated optical tables and complex synchronization of two coherent pulsed laser sources with low noise, matched pulse widths, and precise temporal and spatial overlap. Various fibre-based two-colour pulsed lasers have been proposed for CRS imaging, but limitations such as high cost, low power, and high intensity noise have persisted, necessitating the use of balanced detection schemes to overcome noise issues. Recent advances in fibre optics, including heavy doping of active fibres, double-cladding fibres for efficient pumping, fibre chirped-pulse amplification (FCPA), and multifunctional hybrid fibre components, offer new possibilities for generating high-quality ultrafast laser pulses. Leveraging these advancements, this study presents a novel high-power, low-noise, self-synchronized two-colour pulsed fibre laser system based on coherent wavelength generation (CWG) through cross-phase modulation (XPM). This system aims to enable high-quality CARS and SRS imaging, and other nonlinear imaging modalities without requiring balanced detection, thus increasing imaging speeds while simplifying the detection setup.

The authors review existing literature on coherent Raman scattering (CRS) microscopy and its applications in biomedicine. They highlight the advantages of CRS over fluorescence-based techniques, emphasizing its label-free nature, high resolution, and sensitivity. The limitations of traditional solid-state lasers in CRS microscopy are discussed, focusing on their cost, size, complexity, and the need for vibration isolation. The paper then summarizes previous attempts to develop fiber-based two-color pulsed lasers for CRS, analyzing their respective strengths and weaknesses, and identifying the persistent challenges of high intensity noise and low power, often necessitating the use of balanced detection schemes. The review sets the stage for the introduction of the authors' novel laser design, which aims to overcome these limitations.

The researchers designed a self-synchronized two-colour pulsed fibre laser system employing coherent wavelength generation (CWG) via cross-phase modulation (XPM). The system begins with a passively mode-locked fs fibre laser at 1.0 µm (master laser), generating a pulse train amplified using an FCPA scheme. The output is split into two branches: one for the Stokes beam, amplified using a double-cladding YDFA, and another for the pump beam. The Stokes beam undergoes intensity modulation. The pump beam is generated using the CWG oscillator, which is seeded by a portion of the master laser's output and utilizes an erbium-doped fibre. This passive self-stabilization scheme ensures synchronization between the two beams. The 1.5 µm output of the CWG oscillator is amplified by a DC-EDFA and then frequency-doubled to visible wavelengths using SHG in a PPLN crystal. The spectral characteristics of the laser, including tuning range and pulse width, are thoroughly characterized. The system's noise performance is also evaluated through measurements of power stability, timing jitter using SFG, and relative intensity noise (RIN), comparing it to standard solid-state and SC-based fibre lasers. Finally, the combined beams are integrated into a custom-built laser scanning microscope to acquire CARS and SRS images of biological samples, including living human cells and mouse tissue sections. Detailed experimental setup and simulation results for self-synchronization are provided in supplementary materials.

The developed self-synchronized two-colour pulsed fibre laser demonstrates significant improvements over previous fibre-based systems. The intensity stability is enhanced by 50 dB, the timing jitter is reduced to 24.3 fs, and average power fluctuation is less than 0.5%. The modulation depth exceeds 20 dB, and pulse width variation is limited to 1.8% across the tuning range (2700-3550 cm⁻¹). These characteristics enable high-contrast, fast CRS imaging without the need for balanced detection. The laser's broad tuning range is demonstrated by acquiring SRS spectra of DMSO and methanol, matching spontaneous Raman spectra. Real-time pulse train analysis reveals uniform intensity over long durations. The high modulation depth (over 99%) is confirmed through RF spectrum measurements (SNR of 67 dB at 20 MHz), highlighting the suitability of this all-fibre modulation scheme for lock-in detection. Pulse width measurements reveal values of approximately 2.7 ps (Stokes) and 3.2 ps (pump), maintaining consistency across the tuning range. Long-term power stability measurements over 100 min show fluctuations of only 0.1% and 0.5% for the pump and Stokes beams, respectively. The system's RIN is comparable to standard solid-state lasers at the imaging modulation frequency (20 MHz). High-quality CARS and SRS images of living human cells and mouse tissues were successfully acquired, demonstrating the practical application of the novel laser system for biomedical imaging. The compact size of the entire system (~80 × 85 cm²) is also highlighted.

The results demonstrate the successful development and implementation of a high-performance, compact, and cost-effective two-colour pulsed fibre laser for CRS microscopy. The elimination of the need for balanced detection represents a significant advancement, simplifying the system and increasing imaging speed. The high-quality images obtained from diverse biological samples validate the laser's capabilities for biomedical applications. The improved performance parameters, particularly the significantly reduced noise and stable pulse characteristics, address the major limitations of previous fibre-based CRS systems. This work opens new avenues for the wider adoption and application of CRS imaging techniques in various biomedical research and clinical settings.

This paper presents a novel self-synchronized two-colour pulsed fibre laser system that overcomes the limitations of previous fibre-based lasers for CRS microscopy. The superior performance characteristics, including low noise and high stability, enable high-contrast, fast imaging without balanced detection. The system's compact size and cost-effectiveness promote wider applicability of CRS imaging in biomedicine. Future research could focus on further noise reduction using electronic feedback, optimization of the SHG efficiency for higher pump beam power, and exploration of additional nonlinear imaging modalities.

While the laser system shows significant improvements compared to previous fibre-based systems, the intensity noise level is still slightly higher than that of standard solid-state lasers. This could potentially be further improved through the implementation of electronic feedback control mechanisms. The current design and testing primarily focused on the CH stretching region; further optimization for other spectral ranges might be needed depending on the specific applications. The study primarily focused on specific biological samples; further validation with a wider range of samples would strengthen the generalizability of the findings.