Miniaturized Multicolor Femtosecond Laser Based on Quartz-Encapsulated Nonlinear Frequency Conversion
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis paper introduces a solid-like, quartz-encapsulated femtosecond laser source and demonstrates its advanced biophotonic applications. The research is meaningful, and I suggest it can be published after minor revisions.
- The author needs to explain the advantages of femtosecond lasers at 740 nanometers and 820 nanometers in two-photon microscopy systems.
- Why demonstrate imaging performance through pollen and kidney structure, and what are the details of these materials? Or what representativeness they have, which needs to be explained in the paper.
- Suggest adjusting the structure of the article and consolidating the characterization methods used in the third section into the second section.
- The relevant test results need to add error bars.
- Please increase the resolution of the images in the paper, they are currently too blurry.
Author Response
Comment1:[The author needs to explain the advantages of femtosecond lasers at 740 nanometers and 820 nanometers in two-photon microscopy systems.]
Response1:[We appreciate the reviewer’s comment and the opportunity to clarify the rationale for selecting femtosecond laser excitation wavelengths of 740 nm and 820 nm in our two-photon microscopy system.
In two-photon excitation fluorescence (TPEF) microscopy, these wavelengths are critical for the selective imaging of two key metabolic coenzymes: reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). NADH and FAD are intrinsic fluorophores whose fluorescence provides information on cellular metabolic states. While a single excitation wavelength (e.g., 780 nm) can excite both fluorophores, this approach cannot spectrally separate their signals, making it difficult to perform accurate metabolic quantification. The excitation efficiency of NADH decreases sharply with increasing wavelength in the 700–800 nm range, and its optimal two-photon excitation is generally between 700 and 720 nm. However, for morphological diagnosis, the second harmonic generation (SHG) signal from collagen is also essential. At an excitation wavelength of 720 nm, the SHG signal is centered at 360 nm, which is near the short-wavelength transmission cutoff (360–370 nm) of most glass materials used in objective lens design. This imposes severe constraints on optical design and can degrade image quality. Increasing the excitation wavelength to 740 nm shifts the SHG signal to >370 nm, allowing full transmission through standard microscope objectives without compromising material selection. Considering both metabolic contrast and SHG imaging requirements, 740 nm was determined to be the optimal excitation wavelength for NADH. FAD, in contrast, exhibits an excitation peak near 700 nm but maintains a usable two-photon absorption cross-section in the 800–900 nm range. To isolate FAD excitation from NADH and avoid spectral overlaps, the excitation wavelength must be above 800 nm. At 820 nm, the two-photon absorption cross-section of FAD becomes over 20× greater than that of NADH, rendering NADH fluorescence negligible and enabling highly selective FAD imaging. Using a 500 nm long pass filter in this configuration ensures collection of FAD fluorescence only. Conversely, at 740 nm with a 450 nm short pass filter, NADH fluorescence is effectively isolated.
740 nm: Optimal NADH excitation while ensuring efficient SHG collection for structural imaging.
820 nm: Highly selective FAD excitation with negligible NADH contribution.
This dual-wavelength scheme allows for independent excitation and detection of NADH and FAD, enabling accurate label-free metabolic imaging and simultaneous structural visualization within the same two-photon microscopy platform.]
Comment2:[Why demonstrate imaging performance through pollen and kidney structure, and what are the details of these materials? Or what representativeness they have, which needs to be explained in the paper.]
Response2:[We appreciate the reviewer’s request for clarification regarding our choice of imaging samples and their representativeness.
Pollen grains are commonly used in two-photon and fluorescence microscopy system validation due to their strong intrinsic autofluorescence and wide excitation bandwidth. Their well-defined surface microstructures make them ideal for verifying the core imaging performance of the system—confirming effective light source excitation, optical alignment, and detection sensitivity—without the need for external labels.
Mouse kidney tissue, particularly the glomeruli and renal tubules, provides a biologically relevant structural benchmark for evaluating the high-resolution imaging capabilities of the two-photon microscopy system. Using 740 nm excitation, the system can effectively detect structural information from intrinsic autofluorescence, allowing clear visualization of fine microstructures. Meanwhile, 820 nm excitation combined with a 410 nm filter enables rapid detection of the second harmonic generation (SHG) signal, which originates from collagen fibers. This dual-wavelength imaging approach not only demonstrates the system’s ability to resolve both autofluorescence and SHG signals but also provides a biologically meaningful example: the detection of collagen fibers in kidney tissue is directly relevant for the early diagnosis of renal fibrosis. Therefore, imaging mouse kidney tissue both validates the laser and optical system performance and illustrates a practical biomedical application of the system, highlighting its potential utility in metabolic and structural tissue studies.]
Comment3:[Suggest adjusting the structure of the article and consolidating the characterization methods used in the third section into the second section.]
Response3:[We sincerely appreciate the reviewer’s valuable suggestion regarding the structure of the manuscript.
In the second section, we provide a general description of the system setup and the nonlinear conversion quartz packaging method, which we consider to be a central aspect of the work and serves as an overall framework for the article. The third section, in contrast, is intended to present a more detailed account of the pulse evolution process of the laser. We believe that maintaining this structure allows the manuscript to clearly distinguish between the core system design and the subsequent pulse characterization, facilitating reader understanding. Nevertheless, we are grateful for the reviewer’s insight and have carefully considered the suggestion. We hope that this organizational choice, which separates the system setup from the detailed pulse evolution discussion, provides clarity without compromising the logical flow of the manuscript.]
Comment4:[The relevant test results need to add error bars.]
Response4:[We sincerely thank the reviewer for this suggestion. The data presented in the manuscript were directly measured using a high-precision spectrometer and autocorrelator, and therefore represent individual, accurate measurements rather than statistical datasets. As such, no error bars are included, since the results do not involve repeated trials or statistical analysis. We appreciate the reviewer’s attention to detail and will clarify in the revised manuscript that the presented values correspond to direct instrument measurements.]
Comment5:[Please increase the resolution of the images in the paper, they are currently too blurry]
Response5:[We sincerely thank the reviewer for pointing out the issue with the image clarity. The blurriness was due to an error during the PDF conversion process. We have corrected this and re-uploaded the manuscript with all images provided as high-definition vector TIFF files, ensuring that the figures now meet the expected resolution and quality standards.]
Reviewer 2 Report
Comments and Suggestions for AuthorsThis paper presents an interesting and potentially valuable contribution to the field of miniaturized femtosecond laser sources for two-photon microscopy applications. The authors demonstrate notable innovations including a robust all-solid-state quartz encapsulation process that addresses long-term stability challenges, and a room-temperature quasi-phase-matching design that eliminates the need for active thermal management. The dual-wavelength output at 740 nm and 820 nm targeting specific biological fluorophores (NADH and FAD) represents a thoughtful approach to metabolic imaging applications. The successful demonstration of imaging capabilities on both pollen samples and mouse kidney sections validates the practical utility of the system.
However, several aspects of the work require further clarification and improvement before publication.
1. Physical dimensions of the “miniaturized” system
Given that miniaturization is emphasized as a key advantage in the title, the manuscript lacks essential dimensional specifications. While Figure 1(a) shows the overall system configuration, the authors should provide comprehensive size measurements of the complete system envelope. More critically, Figure 1(b) displaying the quartz-encapsulated structure needs objective dimensional calibration with ruler markings or scale bars to allow readers to assess the actual miniaturization achievement. Without these specifications, claims of miniaturization cannot be properly evaluated.
2. Comparative cost, size, and maintenance analysis
The manuscript would benefit significantly from a systematic comparison of construction costs, integration dimensions, and maintenance requirements relative to conventional Ti:sapphire systems. This could be presented either through detailed textual discussion or a comparative table, as such practical considerations are crucial for potential adopters of the technology.
3. The claim of "eliminating additional active thermal managements"
Regarding the claimed elimination of thermal management systems, the authors attribute this achievement to their optimized room-temperature quasi-phase-matching design using a 19.7 μm poling period in the MgO:PPLN crystal. However, this may be more accurately attributed to the relatively low overall power levels of the system rather than a fundamental breakthrough in phase-matching technology. The authors should consider more nuanced language when describing this aspect, acknowledging that the thermal management elimination may be primarily due to the low-power operation regime rather than an inherent advantage of their phase-matching approach.
4. More details about the pulse duration
The pulse duration characterization requires more comprehensive detail throughout the system. While the authors provide the compressed pulse width of 52.4 fs at 1560 nm and post-frequency-doubling width of 95 fs, they omit the initial pulse width at the oscillator output before pre-chirp management. The statement that "A 4.6-m PM fiber was employed to pre-manage the optical chirp of the seed laser pulses" suggests initial pulse broadening, but quantitative values are missing. This information is essential for understanding the complete pulse evolution through the system.
5. Power stability related
An apparent inconsistency exists in the power stability measurements that requires explanation. The authors report RMS power stability of 0.19% for the 347-mW amplified 1560-nm output, yet claim improved stability of 0.17% after the SPM-based spectral broadening process. This improvement seems counterintuitive, as nonlinear processes typically introduce additional noise rather than reducing it. The authors should provide a detailed analysis of this phenomenon, including potential measurement uncertainties and the physical mechanisms that might account for this apparent stability enhancement. Additionally, stability measurements for the seed oscillator should be included for completeness.
6. Figure quality related
Finally, the overall image quality throughout the manuscript needs substantial improvement. Many figures suffer from poor resolution and clarity, which detracts from the professional presentation of the work and makes it difficult for readers to properly assess the experimental results and system configuration.
Despite these concerns, the fundamental approach and results show promise for advancing compact laser sources for biophotonics applications. With the suggested revisions addressing dimensional specifications, cost analysis, more precise technical claims, comprehensive pulse characterization, stability measurement clarification, and improved figure quality, this work could make a solid contribution to the field.
Author Response
Comment1:[Physical dimensions of the “miniaturized” system. Given that miniaturization is emphasized as a key advantage in the title, the manuscript lacks essential dimensional specifications. While Figure 1(a) shows the overall system configuration, the authors should provide comprehensive size measurements of the complete system envelope. More critically, Figure 1(b) displaying the quartz-encapsulated structure needs objective dimensional calibration with ruler markings or scale bars to allow readers to assess the actual miniaturization achievement. Without these specifications, claims of miniaturization cannot be properly evaluated]
Response1:[We thank the reviewer for this valuable suggestion. We have measured the dimensions of the quartz-encapsulated structure, and the total length is 52.2 mm. A scale bar has now been added to Figure 1(b) to provide clear dimensional reference. This addition will allow readers to more accurately assess the degree of miniaturization achieved in our design.]
Comment2:[The manuscript would benefit significantly from a systematic comparison of construction costs, integration dimensions, and maintenance requirements relative to conventional Ti:sapphire systems. This could be presented either through detailed textual discussion or a comparative table, as such practical considerations are crucial for potential adopters of the technology.]
Response2:[We appreciate the reviewer’s constructive suggestion. In response, we have added a systematic comparison between conventional Ti:sapphire laser systems and the fiber-laser-based system presented in this work, covering construction cost, integration dimensions, and maintenance requirements.
From the perspectives of size, weight, structural layout, and operational performance, our fiber-laser system offers distinct advantages over Ti:sapphire systems. Its ultra-compact and lightweight design is well-suited for portable or space-constrained environments, while the sealed and robust architecture provides strong resistance to dust, vibration, and temperature fluctuations. The system operates in an alignment-free manner, enabling direct fiber output for easy integration with experimental setups, and maintains high output stability over long periods without daily optimization. In addition, the architecture is inherently scalable, allowing adaptation to higher pulse energies or specific repetition rates as needed. These features collectively reduce maintenance complexity and cost.]
Comment3:[The claim of "eliminating additional active thermal managements"
Regarding the claimed elimination of thermal management systems, the authors attribute this achievement to their optimized room-temperature quasi-phase-matching design using a 19.7 μm poling period in the MgO:PPLN crystal. However, this may be more accurately attributed to the relatively low overall power levels of the system rather than a fundamental breakthrough in phase-matching technology. The authors should consider more nuanced language when describing this aspect, acknowledging that the thermal management elimination may be primarily due to the low-power operation regime rather than an inherent advantage of their phase-matching approach.]
Response3:[We thank the reviewer for this comment. We agree that the elimination of additional active thermal management in our system is closely related to the relatively low overall average power of the laser, and we have revised the manuscript to use more nuanced language accordingly. In the updated text, we clarify that while the optimized room-temperature quasi-phase-matching design with a 19.7 μm poling period in the MgO:PPLN crystal contributes to stable operation without active thermal control, this benefit is realized in conjunction with the low-power operating regime of the system.( Lines 179-183 are marked in red) The revised description now acknowledges that our phase-matching design is advantageous for simplifying the optical layout and maintaining stability under the given operating conditions, but does not in itself constitute a universal solution for eliminating thermal management in all high-power scenarios. This adjustment reflects a more accurate attribution of the observed performance and aligns with the reviewer’s recommendation.]
Comment4:[The pulse duration characterization requires more comprehensive detail throughout the system. While the authors provide the compressed pulse width of 52.4 fs at 1560 nm and post-frequency-doubling width of 95 fs, they omit the initial pulse width at the oscillator output before pre-chirp management. The statement that "A 4.6-m PM fiber was employed to pre-manage the optical chirp of the seed laser pulses" suggests initial pulse broadening, but quantitative values are missing. This information is essential for understanding the complete pulse evolution through the system.]
Response4:[We thank the reviewer for this valuable comment. In the revised manuscript, we have added the missing quantitative detail on the initial pulse duration at the oscillator output, before entering the pre-amplification stage. The seed laser generated pulses with a full-width at half-maximum (FWHM) duration of 55 fs at 1560 nm.
These values have been added to the revised manuscript (line 87) to provide a complete characterization of the pulse evolution from the oscillator to the final compressed state and post-frequency-doubling stage. This addition ensures that the full temporal progression of the pulse through the system is clearly documented.]
Comment5:[An apparent inconsistency exists in the power stability measurements that requires explanation. The authors report RMS power stability of 0.19% for the 347-mW amplified 1560-nm output, yet claim improved stability of 0.17% after the SPM-based spectral broadening process. This improvement seems counterintuitive, as nonlinear processes typically introduce additional noise rather than reducing it. The authors should provide a detailed analysis of this phenomenon, including potential measurement uncertainties and the physical mechanisms that might account for this apparent stability enhancement. Additionally, stability measurements for the seed oscillator should be included for completeness.]
Response5:[We sincerely thank the reviewer for raising this important point. We note that a very small portion of the power measured in the 1560-nm band originates from amplified spontaneous emission (ASE) in the gain fiber. As the ASE arises from a spontaneous emission process, these components do not participate in the subsequent frequency doubling stage. Consequently, the RMS power measured at 780 nm after the SPM-based spectral broadening and frequency doubling stage is 0.17%, which is slightly lower than the 0.19% RMS measured at 1560 nm. Both values demonstrate the high stability of the system.]
Comment6:[Finally, the overall image quality throughout the manuscript needs substantial improvement. Many figures suffer from poor resolution and clarity, which detracts from the professional presentation of the work and makes it difficult for readers to properly assess the experimental results and system configuration.]
Response6:[We sincerely thank the reviewer for highlighting the issue with image clarity. The reduced resolution in the original submission was caused by an error during the PDF conversion process. This has now been corrected, and the manuscript has been re-uploaded with all figures replaced by high-definition vector-based TIFF files. The updated figures meet professional publication standards in both resolution and clarity, ensuring that the experimental results and system configuration can be accurately assessed by readers.]
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors report on a femtosecond laser encapsulated in quartz with a repetition rate of 80 MHz, which delivers femtosecond laser pulses at 740 nm and 820 nm. According to the authors, this laser system was built in-house using an erbium-doped fiber laser as the fundamental laser source. The frequency conversion SHG and SPM is performed using quartz. Pulses of 30 mW, 53.7 fs at 740 nm and 15 mW, 60.8 fs at 820 nm are achieved. In addition, a two-photon microscope was set up and validated for imaging. This enabled kidney sections from a mouse and pollen samples to be examined by detecting the corresponding fluorescence signals.
The study is easy to understand and, apart from a few formal improvements in the quality proof phase, can be published as it is . The only formal physical comment on the content is the scaling of graphs 2 (ab), 3 (ab) and 5 (ab). These look like normalized curves, but appear to be normalized to 1. If this is the case, normalized would be better than a.u. and, if necessary, a reference to the same 3 subgraphs should be sought in order to better compare the results.
The representation of the pulse width with 52.4 × 1.414 fs would be better with 52.4 · 1.414 fs instead of a cross, as it is a scalar. Some variables are in italics instead of bold (lines 102, 104, 105, 112) and some spaces are missing between variables and values. In line 92, the exponent has slipped.
All graphs appear blurred; a better resolution should be submitted for the final version.
Comments for author File: 
Comments.pdf
Author Response
Comment:[The study is easy to understand and, apart from a few formal improvements in the quality proof phase, can be published as it is . The only formal physical comment on the content is the scaling of graphs 2 (ab), 3 (ab) and 5 (ab). These look like normalized curves, but appear to be normalized to 1. If this is the case, normalized would be better than a.u. and, if necessary, a reference to the same 3 subgraphs should be sought in order to better compare the results.
The representation of the pulse width with 52.4 × 1.414 fs would be better with 52.4 · 1.414 fs instead of a cross, as it is a scalar. Some variables are in italics instead of bold (lines 102, 104, 105, 112) and some spaces are missing between variables and values. In line 92, the exponent has slipped.
All graphs appear blurred; a better resolution should be submitted for the final version.]
Response:[We sincerely thank the reviewer for the careful reading and valuable comments. Regarding the figures, the curves in graphs 2 (a, b), 3 (a, b), and 5 (a, b) are normalized to facilitate comparison of their shapes. We have kept the labeling as “a.u.”, as it accurately reflects the nature of the data while maintaining consistency with common practice in the field.
Concerning the pulse width representation, the “×” symbol in 52.4 × 1.414 fs indicates scalar multiplication in the geometric operation rather than vector multiplication. We have retained this notation, as it is widely used and clearly understood in the laser and photonics community.
We are very grateful for pointing out other formatting issues, including variable styles, missing spaces, and the exponent in line 92, and all these have been carefully corrected in the revised manuscript. Additionally, all graphs have been replaced with higher-resolution versions to ensure clarity and meet publication standards.]
