Miniaturized Multicolor Femtosecond Laser Based on Quartz-Encapsulated Nonlinear Frequency Conversion

Abstract

Ultrafast lasers operating at 740 nm and 820 nm have attracted widespread attention as two-photon light sources for the detection of biological metabolism. Here, we report on a solid-like quartz-encapsulated femtosecond laser with a repetition rate of 80 MHz, delivering 740 nm and 820 nm femtosecond laser pulses. This home-built laser system was realized by employing an erbium-doped 1560 nm fiber laser as the fundamental laser source. A quartz-encapsulated nonlinear frequency conversion stage, consisting of a second-harmonic generation (SHG) stage and self-phase modulation (SPM)-based nonlinear spectral broadening stage, was utilized to deliver 30 mW, 53.7 fs, 740 nm laser pulses and the 15 mW, 60.8 fs, 820 nm laser pulses. Further imaging capabilities of both wavelengths were validated using a custom-built inverted two-photon microscope. Clear imaging results were obtained from mouse kidney sections and pollen samples by collecting the corresponding fluorescence signals. The achieved results demonstrate the great potential of this laser source for advanced two-photon microscopy in metabolic detection.

1. Introduction

Two-photon microscopy (TPM), widely recognized as one of the most effective optical techniques for real-time, in vivo, and non-invasive imaging of live tissues and biological systems, has found broad applications across diverse biomedical fields, such as brain imaging [1,2], endoscopy [3,4], intravital pathology [5,6], and dermatological diagnostics [7,8]. TPM induces two-photon absorption of endogenous fluorophores within cells, enabling label-free imaging of cellular structures and metabolic activity using femtosecond excitation wavelengths between 700 and 900 nm [9,10]. In particular, two-photon excitation of the intrinsic cofactors flavin adenine dinucleotide (FAD, efficiently excited at 820 nm) and nicotinamide adenine dinucleotide (NADH, efficiently excited at 740 nm) allows for simultaneous acquisition of both structural and metabolic information of biological tissues [11,12], which is especially valuable for applications such as cancer diagnostics [13] and metabolic profiling [14]. Therefore, the femtosecond lasers operating at both 740 nm and 820 nm can efficiently excite these endogenous fluorophores and improve the accuracy and efficiency of the non-invasive in vivo cellular detection [15].

For the past decades, femtosecond Ti:sapphire lasers have been the generally utilized light sources for two-photon imaging systems, delivering <100 fs pulses with the wavelength range spanning from 700 nm to 1100 nm [16,17]. However, the complex systematic construction and high costs of Ti:sapphire lasers limit their further practical applications in advanced biomedical research [18]. Fiber lasers have attracted increasing attention due to their structural stability and simple systematic design [19]. L. Huang et al. demonstrated a 61 mW, 80 fs, 790 nm laser source employing the SHG with an Er-doped 1560 nm fiber laser, subsequently utilized to realize the imaging of the femurs of mice and pigs [11]. L.T. Chou et al. employed a 48 MHz, 120 fs, ytterbium-doped, 1 µm fiber laser to produce 740 nm pulses with an average power of 91.2 mW with the SPM [20]. This 740 nm femtosecond laser was further utilized to illuminate mouse MCF-7 breast tumor cells inside the TPM. The complexity of fiber laser systems for autofluorescence applications, which leads to long-term instability, hinders their practical use. Addressing these issues, the SHG of the 1560 nm nonlinearly amplified femtosecond laser and the SPM of the frequency-doubled 780 nm femtosecond laser can be quartz-encapsulated together in generating 740 nm and 820 nm femtosecond lasers. To our knowledge, this kind of femtosecond laser delivering both 740 nm and 820 nm laser contents is rarely reported. Furthermore, this design features high power stability and a compact packaging structure, enabling a plug-and-play connection between the laser source and the imaging probe. Therefore, an efficient two-photon excitation can be achieved, facilitating the acquisition of both morphological and metabolic information from tissues and cells.

In this study, we report on a solid-like, quartz-encapsulated femtosecond laser source with a repetition rate of 80 MHz, delivering dual-wavelength pulses at 740 nm and 820 nm. The system originates from a fully polarization maintenance (PM) Er-doped fiber laser operating at 1560 nm, which produces 52.4 fs pulses. These fundamental pulses are first frequency-doubled to 780 nm in a periodically poled lithium niobate (PPLN) crystal. The resulting light is then coupled into a photonic crystal fiber (NL-PM750) for spectral broadening via supercontinuum generation. To ensure exceptional long-term stability, all nonlinear optical components are integrated and sealed within a robust quartz module. This compact and highly reliable source simultaneously provides two distinct outputs: 53.7 fs pulses at 740 nm with an average power of 30 mW and 60.8 fs pulses at 820 nm with an average power of 15 mW, making it highly suitable for applications such as two-photon microscopy.

2. Laser Setup and Quartz-Encapsulated Structure

Figure 1a illustrates the experimental configuration of the frequency-doubled Er-doped fiber laser system. The seed laser is an 80 MHz mode-locked Er-doped fiber oscillator utilizing a nonlinear amplifying loop mirror (NALM), delivering pulses with a duration of 55 fs [21,22]. A 4.6 m PM fiber (PM1550, Coherent, Saxonburg, PA, USA) was employed to pre-manage the optical chirp of the seed laser pulses. The all-PM fiber amplifier consisted of a PM wavelength division multiplexer, two 850 mW 976 nm single-mode laser diodes, and a 3.6 m Er-doped gain fiber (DHB1500, Fibercore, Southampton, UK). It was utilized to scale up the average power of the seed laser from 2.9 mW to 350 mW. The combination of the Er-doped gain fiber (29.2 fs²/mm) and the PM1550 (−23.5 fs²/mm) spliced before and after the gain fiber was utilized to enable the nonlinear amplification and compression processes [23]. Based on contributions of the dispersion management and the self-phase modulation, the optical pulse duration of the nonlinearly amplified 357 mW 1560 nm laser pulses was nonlinearly compressed to 52.4 fs based on the Gaussian assumption, directly delivered from the fiber output port.

The 357 mW, 80 MHz laser pulses were further delivered into a quartz-encapsulated module to realize the optical collimation, optical isolation, frequency doubling, polarization management, and nonlinear spectral broadening. Figure 1b illustrates the real picture of the quartz-encapsulated module. The 357 mW, 1560 nm laser beam was firstly collimated with an aspheric lens L1 (f = 11 mm, 354064C, Lightpath, Orlando, FL, USA). The corresponding collimated beam diameter was 2.7 mm. A combination of a half waveplate, a focusing aspheric lens L2 (f = 6.2 mm, 354171C, LightPath, Orlando, FL, USA), a MgO:PPLN (C-M-1940-S-1, Foctek Photonics, Fuzhou, China) nonlinear crystal, and a collimating aspheric lens L3 (f = 6.2 mm, 354171B, LightPath, Orlando, FL, USA) was utilized to realize the SHG of the 1560 nm femtosecond laser. The poling period of 19.7 μm of the 0.5 mm long MgO:PPLN crystal was employed in realizing the nonlinear phase matching condition. The optimal quasi-phase-matching temperature of the crystal was designed to be room temperature, eliminating additional active thermal management. The generated 148 mW, 80 MHz, 780 nm frequency-doubled laser pulses were further coupled into a high nonlinearity photonic crystal fiber (HNPCF, NL-PM-750, NKT Photonics, Blokken, Birkerød, Denmark) with an aspheric lens L4 (f = 1.45 mm, 354140B, LightPath, Orlando, FL, USA) to realize the nonlinear spectral broadening. The NL-PM-750 photonic crystal fiber with a tightly confined mode-field diameter of approximately 1.6 μm, featuring near-zero dispersion at 750 nm and a group velocity dispersion (GVD) parameter of β₂ = −0.0066 ps²/m at 780 nm, enabled the efficient SPM-induced nonlinear spectral broadening process over 2 cm HNPCF. The realized coupling efficiency was measured to be approximately 70% with a coupled pulse energy of around 1.25 nJ, indicating a perfect optical coupling construction.

Realizing the quartz-encapsulated module, the utilized aspheric lenses, waveplates, free-space isolator, and nonlinear crystal were carefully encapsulated inside the quartz glass tubes with identical outer diameters. To achieve structural integration and stable coupling, the HNPCF was firstly collapsed at the input port and then inserted into a quartz glass sleeve for further protection. A quartz glass tube with the same outer diameter as the fiber’s quartz sleeve was subsequently bonded to the rear side of the coupling lens (L4), ensuring that the fiber end face was precisely positioned at the focal plane of the lens. To further enhance the alignment accuracy and bonding strength between the photonic crystal fiber (encapsulated in the quartz sleeve) and the coupling lens, an additional quartz glass tube was employed to connect the two components, thereby reinforcing mechanical stability and enabling integrated assembly. All optical elements were precisely aligned and fixed using UV-curable optical adhesives. Specifically, the end-face bonding of quartz tubes was performed using EM10483 (EMIUV, Breckenridge, CO, USA), a high-glass transition temperature adhesive that offers excellent thermal and mechanical stability. For the interface between the optical components and the inner wall of the quartz housing, NOA61 (Norland, Jamesburg, NJ, USA) was selected for its superior fluidity, which promotes better wetting and increases the effective bonding area. UV curing was conducted under controlled conditions to avoid thermal stress and ensure stable alignment. This hybrid bonding strategy enabled the rigid, low-shrinkage encapsulation of all components, suppressing vibration- and temperature-induced misalignments. The overall packaging approach ensured precise optical alignment and robust mechanical integration of the SHG and frequency-shift modules, thereby enhancing the long-term stability and reliability of the output femtosecond pulses.

3. Results

Figure 2a illustrates the optical spectrum of the nonlinearly compressed 347 mW, 80 MHz, 1560 nm signal pulses delivered by the output port of the PM1550 compression fiber. The measured 3 dB spectral bandwidth and 10 dB bandwidth were 3 nm and 64 nm, respectively. Figure 2b presents the measured autocorrelation trace of the compressed pulses. The corresponding pulse duration was estimated to be 52.4 × 1.414 fs based on the Gaussian assumption. Notably, the trace exhibits pronounced pedestals, which originate from uncompensated high-order phase components—primarily attributed to residual third-order dispersion. Figure 2c plots the measured optical pulse train with the repetition rate of 80 MHz, indicating a stable mode-locking operation state of the 1560 nm fiber oscillator. Figure 2d plots the measured root-mean-square (RMS) of 0.19% @1h of the delivered 347 mW nonlinearly amplified 1560 nm laser pulses under the coupled pump power of 1.7 W. The output power was monitored with a high-resolution power meter (S314C) (Thorlabs, Newton, MA, USA) with a resolution of 5 µW. Through careful dispersion and nonlinear phase management, a femtosecond fiber laser output was ultimately achieved, delivering 55 fs pulses at an 80 MHz repetition rate with an average power of 347 mW. This output served as the pump source for subsequent nonlinear frequency conversion processes.

Figure 3a illustrates the measured optical spectrum of the SHG pulses, centered at 780 nm with a full width at half maximum (FWHM) of 7 nm. Figure 3b shows the corresponding autocorrelation trace, and the measured pulse duration was estimated to be 95 × 1.414 fs, assuming a Gaussian profile. As shown in Figure 3d, the calculation of the phase-matching condition reveals that the optimal poling period for the PPLN crystal is 19.7 μm at a temperature of 300 K. The frequency doubling process yielded an average output power of 145 mW, corresponding to a conversion efficiency of 40.3%. Through appropriate selection and design, the quasi-phase-matching temperature of the frequency-doubling crystal was set to room temperature. In the context of biological imaging applications, the combination of low average power and the absence of active thermal control was identified as one of the contributing factors to the high stability of the system. A third aspheric lens (354171B, LightPath, Orlando, FL, USA) was employed after the PPLN crystal to collimate the frequency-doubled output beam. The resulting output beam profile is shown in Figure 3c, exhibiting a beam diameter of 1.45 mm and an ellipticity of 0.95, indicating good spatial quality. Furthermore, one-hour power stabil Orlando, USA ity measurements were performed. The RMS of the power fluctuation was measured to be 0.17%, demonstrating excellent long-term stability of the SHG femtosecond output.

Figure 4a illustrates the dispersion and loss parameters of the HNPCF; the near-zero dispersion point of this HNPCF is around 750 nm. Figure 4b shows the output spectrum after nonlinear broadening. A distinct dual-peak structure is observed, with the left peak centered at 740 nm and the right peak centered at 820 nm. The short-wavelength peak arises primarily from blue-shifted frequency components at the leading edge of the pulse, while the long-wavelength peak originates from red-shifted components at the trailing edge.

After collimation of the output beam using an aspheric lens L5 (354140B, LightPath, Orlando, FL, USA), spectral filtering was performed using a 760 nm short-pass filter (SP760, JiuShi Optics, Shanghai, China). The resulting output pulse exhibited an average power of 30 mW. The corresponding filtered spectrum is shown in Figure 5a, with a central wavelength of 741 nm. The autocorrelation trace of the output pulse is presented in Figure 5b, and the pulse duration, assuming a Gaussian temporal profile, was measured to be 53.7 × 1.414 fs. Subsequently, spectral filtering was also carried out using an 800 nm long-pass filter (LP800, JiuShi Optics, Shanghai, China). The filtered output exhibited an average pulse power of 15 mW. The corresponding spectrum, with a central wavelength of 820 nm, is depicted in Figure 5c. The autocorrelation trace of this signal is shown in Figure 5d, yielding a pulse duration of 60.8 × 1.414 fs under the Gaussian assumption.

The 740 nm and 820 nm femtosecond pulses were further routed to a miniaturized multiphoton microscopy probe to evaluate its imaging performance. The imaging setup is shown in Figure 6a. In this setup, the excitation light path is marked in red and the collection light path is marked in green. After passing through the filter, the collimated beam was directly coupled into the probe without the need for anti-resonant fibers or photonic crystal fibers. The inverted two-photon microscope used for imaging validation consists of a Micro-Electro-Mechanical System (MEMS) scanning mirror, a scan lens, a dichroic mirror, an objective lens, and a condenser lens, as shown in Figure 6a. The MEMS scanner operates at a frequency of 2400 Hz, with a frame rate of 3 fps and a field of view of 300 µm × 300 µm. The excitation light is focused by a 9× microscope objective (N.A. = 0.7). The generated harmonic signals are collected by the same objective, collimated by a collimating lens into a parallel beam, and then spectrally separated by the dichroic mirror to acquire fluorescence signals in two separate channels. These signals are subsequently directed by the condenser lens onto the detection surface of a photomultiplier tube (PMT).

Figure 6c presents an image of pollen acquired using 10 mW excitation at 740 nm, clearly revealing the fine structural details. Under the same excitation power, imaging with 820 nm pulses (Figure 6d) also delineates distinct pollen features. To further evaluate the imaging performance of our dual-wavelength femtosecond laser, we performed two-photon imaging of mouse fibrotic kidney sections. Firstly, autofluorescence from the tissue was excited using the 740 nm output, and the signal was collected through a 390 nm long-pass filter, revealing key renal structures in a green pseudo color (Figure 6e). The resulting images clearly resolved the glomeruli and surrounding renal tubules. Subsequently, the sample was imaged using the 820 nm output to excite SHG from collagen fibers. The SHG signal was isolated with a 410 nm bandpass filter, visualizing the collagen distribution in a red pseudo color (Figure 6e). Thus, the 740 nm channel was used to observe the glomerular architecture, while the 820 nm channel targeted collagen in the fibrotic kidney, demonstrating the system’s capability for multimodal imaging.

4. Discussion

The demonstrated quartz-encapsulated femtosecond fiber laser provides a compact, stable, and versatile excitation source for two-photon microscopy. By delivering femtosecond pulses at 740 nm and 820 nm, the system directly covers the optimal two-photon excitation ranges of two major endogenous fluorophores involved in cellular metabolism (NADH and FAD), thereby enabling label-free metabolic imaging. The successful imaging of pollen grains and mouse kidney sections validates not only the optical performance of the laser but also its suitability for resolving biologically relevant structures at subcellular scales. These results suggest that, in specific non-laboratory application scenarios, such as portable [24] or space-constrained imaging environments [25,26], this quartz-encapsulated femtosecond fiber laser offers distinct advantages over conventional Ti:sapphire systems, including compactness, robustness, and reduced maintenance requirements.

In addition to the proof-of-concept demonstration, our study highlights several potential application scenarios for two-photon biological imaging. The dual-wavelength design is particularly well suited for optical redox ratio measurements, which are increasingly recognized as a powerful tool for assessing cellular metabolic states and detecting early pathological changes, such as cancer progression or tissue hypoxia [27,28,29]. By enabling selective excitation of NADH and FAD at their respective optimal wavelengths, the system enhances the accuracy of metabolic imaging compared with single-wavelength approaches. The collagen-derived second-harmonic generation signal detected at 820 nm highlights the potential of this light source for investigating extracellular matrix remodeling at both the cellular and tissue levels [30,31]. Quartz encapsulation further enhances the system by ensuring mechanical robustness, environmental stability, and long-term alignment-free operation. These features are crucial for enabling in vivo animal imaging and clinical endoscopic physiological monitoring [32].

Future research could focus on expanding the system to achieve higher pulse energies or adjustable repetition rates, which would enable multi-parameter imaging and the simultaneous multi-photon excitation of additional endogenous or exogenous fluorophores. Such enhancements would broaden the versatility of the platform, allowing more comprehensive metabolic and structural studies across diverse biological samples and experimental conditions.

5. Conclusions

In this study, we report on a solid-like, quartz-encapsulated femtosecond laser source, with a repetition rate of 80 MHz, delivering synchronous dual-wavelength pulses at 740 nm and 820 nm. The systematic long-term stability and robustness were realized by encapsulating the employed free-space optics within a sealed quartz module. Based on the home-built 80 MHz, 347 mW, 52.4 fs, 1560 nm laser pulses, the 80 MHz, 30 mW, 53.7 fs, and 740 nm laser pulses and 80 MHz, 15 mW, 60.8 fs, and 820 nm laser pulses can be delivered by this quartz-encapsulated module. The measured RMS of the output power was 0.17%@1h, indicating a perfect operating performance. We have validated the practical utility of this source by conducting two-photon imaging on mouse kidney sections and pollen samples, successfully resolving distinct biological structures through autofluorescence and second-harmonic generation. The results demonstrate that this compact and highly stable laser system is a powerful and versatile tool for advanced biophotonic applications, particularly for multimodal two-photon microscopy in metabolic studies.