Frame-Stacking Method for Dark Digital Holographic Microscopy to Acquire 3D Profiles in a Low-Power Laser Environment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper presents a 3D holographic imaging method under low laser illumination, which appears to be effective. The result is quite meaningful, and the experimental results are sufficiently presented. I recommend acceptance after minor revisions.

1. Polarization information can also be used in digital holography. It could be briefly introduced in the introduction, and relevant papers should be cited: APL Photonics, 2023, 8(7); Optics Letters, 2024, 49 (7), 1696-1699.
2. Figures 6 and 2 are too similar; it is recommended to modify or merge them.
3. Figure 9 uses a direct screenshot from the program, which is unprofessional. It is recommended to modify it.
4. It is suggested to combine Tables 1-4 into a single device parameter table.
5. How can the effectiveness be validated through reconstruction accuracy or error metrics? It is recommended to add some quantitative indicators to evaluate the advantages of the proposed method. For example, in image enhancement, there are both subjective and objective metrics. You may refer to: Information Fusion 2025, 117, 102809; Photonics 2024, 11 (8), 773.

 

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

Holographic imaging method is introduced. 

How it compare with resolution and simplicity of implementation when compared with non-coherent holography (Roadmap on computational methods in optical imaging and holography

J Rosen, S Alford, B Allan, V Anand, S Arnon, FG Arockiaraj, J Art, B Bai, ...

Applied Physics B 130 (9), 166)

Presence of speckles is expected to cause issues

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

The paper introduces a method aiming to improve the accuracy of 3D profiles obtained from Digital Holographic Microscopy (DHM) in low-power laser environments. The proposed method uses image decomposition, superimposition, and rescaling to reduces noise and enhance signal quality. Experimental validation is presented using both synthetic and real biological samples (e.g., red blood cells).

While the topic is relevant to DHM and its applications in medical and biological imaging, the approach is largely based on existing methods, and offers incremental rather than transformative advancements.

1. Lack of Novelty:
• Capturing low-intensity holograms is a well-established area of research. Techniques such as frame averaging, HDR imaging, and noise reduction have been extensively studied in the literature. Seed the following Refs.
• The main idea—combining frame superimposition and rescaling—appears incremental and does not significantly advance the state of the art.

i.       Masaki Yamamoto, Hirotsugu Yamamoto, and Yoshio Hayasaki, "Photon-counting digital holography under ultraweak illumination," Opt. Lett. 34, 1081-1083 (2009)

 

ii.     R. YONESAKA, Y. AWATSUJI, T. TAHARA, X. PENG, S. URA, and K. NISHIO, "Digital Holography Using High Dynamic-Range Imaging," in Digital Holography and Three-Dimensional Imaging, OSA Technical Digest (online) (Optica Publishing Group, 2013), paper DTh3A.3.

 

iii.   Yonesaka, R., Lee, Y., Xia, P., Tahara, T., Awatsuji, Y., Nishio, K., & Matoba, O. (2016). High dynamic range digital holography and its demonstration by off-axis configuration. IEEE Transactions on Industrial Informatics, 12(5), 1658-1663.

This means the literature study is not completed and still work to do in the state of the art to adapt the main objective and the hypothesis of the work.

2. Insufficient Benchmarking:
• The manuscript lacks comparisons with other established methods for low-intensity holography, such as photon counting, adaptive exposure techniques, or computational image reconstruction.
• Without benchmarking, it is difficult to assess whether the proposed DHS system outperforms or complements existing techniques.
3. Generalization Issues:
• The system assumes static objects during hologram acquisition, which limits its applicability to dynamic biological samples or environments.

5. Recommendations

Revisions Required Before Publication

1. Strengthen Novelty: Clearly articulate how the proposed DHS system differs from or improves existing low-intensity holography methods.
2. Benchmarking: Include comparisons with state-of-the-art techniques, demonstrating the advantages and limitations of DHS compared to alternative approaches.
3. Expand Generalization: Address the system's applicability to dynamic samples and diverse object types, or explicitly define its scope and limitations.
4. Broaden the Discussion: Integrate the method into the broader context of computational imaging, HDR techniques, or AI-based holography to highlight its potential impact.
5. Technical Depth: Provide more technical insights into how the superimposition and rescaling algorithms work, and explore the trade-offs involved.

If these revisions are implemented, the paper could meaningfully contribute to the field of DHM. However, in its current form, the manuscript does not meet the standards for publication.

Author Response

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Reviewer 4 Report

Comments and Suggestions for Authors

The authors have investigated the effect of low power light illumination on the 3D profile measurement on microscopic transparent phase objects and investigated that height profiles of objects can be accurately reconstructed by a hologram that is a superimposition of several frames (holograms images) from a video recording of holograms images at low power illumination. In this investigation, the authors have used the well-known hologram recording geometry (Michelson interferometer) and a hologram reconstruction method. The reviewer has the main concerns that this method requires a large number of holograms for 3D height profile reconstruction at low-power object illumination and that is why this method is not suitable to study microscopic biological objects like RBCs, as RBCs exhibit height fluctuations with time. The fluctuation parameters of RBCs are related to the physiological state of RBCs so the method of recording several holograms at low illumination cannot be applied for disease diagnostics. This method is applicable/restricted to phase objects that don’t fluctuate in height or fluctuate very slowly. Thus, the limitations of your method should be clearly indicated in the paper along with the possible applications of the proposed method. Apart from the above major concerns, my other comments are appended below –

1.      The title of paper includes a word “Dark Hologram System (DHS)”, this group od words is not appropriate in the title because the experimental system/setup is a well-known Mach-Zehnder interferometer, and no new component/equipment is added in the system also it is widely available in the literature. Change the title of paper accordingly.

2.  The same Mach-Zehnder type experimental setup with two objective lenses in two arms of interferometer is already available in the following references –

(a)   Phase contrast imaging of red blood cells using digital holographic interferometric microscope. In Third International Conference on Applications of Optics and Photonics (SPIE Vol. 10453, pp. 678-684, 2017).

(b)   Characterization of Micro-lenslet Array Using Digital Holographic Interferometric microscope. Optics, Photonics and Laser Technology, 218, 25 (2018).

The above references should be cited in the manuscript to give credit to other researchers who have worked in the related research field.

3. The sentences in the Abstract, “DHM requires several experimental instruments,” the instruments in the sentence should be replaced with components.

4. The sentences in line 71-73, “we investigate the relationship between the number of superimposed videos corresponding to the irradiation time and 3D profiles, as well as the characteristics of the power and 3D profiles,”. In your work, you have used/superimposed the frames of a recoded video of hologram images not the superposition of videos itself. Your sentence is not clear please write the sentence clearly.

5. How was the reference hologram (without object) recorded in the experiments?  In recording the reference holograms did you completely remove the glass slide from the object arm of interferometer or recoded it in different way? Please clarify in the manuscript.

6. The figure captions in the manuscript should be self-explanatory and descriptive. In this regard, revise all the captions of figures. For an example, Caption of Fig. 3 should be: Scheme of wavefront distortion by a transparent phase object with high refractive index.

7.   What was the surrounding medium in case of microsphere imaging and its refractive indexed value? What was the value of refractive index of surrounding medium in case of RBC? What is the value of RBCs in your experiments? Please mention these values in the manuscript.

8.   The sentence in lines 146-148, “This is because when the light enters the image sensor, the light passing through the object and the light after passing through the object are acquired without distinguishing between them”, is not clear. What do mean to say - the light passing through the object and the light after passing through the object are acquired without distinguishing between them. Please write the sentence with clarity. Similarly, the sentence in line 165-167 is not clear. What do you mean by loss of 0?  Write the sentence clearly.

9.  The equations (6) and (7) are not clear. The reviewer thinks that I_HOD is one of the frames from a video recording of holograms images at low power illumination. For the summation of N frames starting from i=1 to i=N in equations (6) and (7) a subscript or superscript of i should be added to I_HOD.

10.  The authors should necessarily add cross-sectional height profiles along the x-axis or y -axis in fig. 14, fig. 15, fig. 17, fig. 19, fig. 20 and fig. 21. For example, in Fig. 14, one more figure containing cross-sectional height profiles say along x-axis, from 3D height profile: Conventional method, DHS, and ideal comparison model should be provided for comparison purpose, and it should be discussed in the manuscript. Similarly, cross-sectional profiles along x-axis at any y pixel position that passes through the diameter of the object should be compared in a new fig. 15 (e): Cross-sectional profiles along the x-axis corresponding to Fig. 15 (a)-(f).

11. How did you reconstruct the ideal 3D shape of the object in Fig. 14? Explain it. What mathematical model did you use to reconstruct this ideal 3D height shape in Fig. 14. Also compare the cross-sectional profiles along x-axis in Fig. 14, 15, 17, 19 with ideal profile.

12.  Add the limitations of your method in the conclusion section.

13. The last sentence in the conclusion part should be modified or clarified how the proposed method can be applied in the medical industry as it takes a lot of time and dynamic characteristics of biological samples cannot be extracted with this method.

The paper can be considered for publication after properly addressing the above concerns.

Comments on the Quality of English Language

At many places, the sentences are not clear. Please write clearly.

Author Response

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Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

accepted

Author Response

Thanks for the review.

Reviewer 3 Report

Comments and Suggestions for Authors

I think the authors addressed most of my comments. Still, I have one left which is related to the phase modulation given in Eq. 2 which is not correctly added. Did the other mean the spatial carrier frequency required to separate the hologram +1, 0, and -1 orders at the Fourier domain? If this is the case, then the phase ramp ø has to be changed to another symbol avoiding any confusion with the phase difference in Eq. 3.

Author Response

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Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed almost all my comments and concerns. Still, the following minor comments are left-

1.  Please remove the high accuracy word from the title. Because the phase reconstruction with your method is not as good as in the case of normal light illumination.
2. There are errors in the reference [29]. The author’s name and journal name are not correct. Also, the title of reference is not complete. Correct ref. is -

Kumar, V.; Shakher, C. (2018). Characterization of Micro-lenslet Array Using Digital Holographic Interferometric Microscope. In: Ribeiro, P., Raposo, M. (eds) Optics, Photonics and Laser Technology. Springer Series in Optical Sciences, vol 218. Springer. https://doi.org/10.1007/978-3-319-98548-0_2

3. The sentence written from line 202-204 should be correct as –

After this processing, the 3D image is converted from a reference image and an object image, achieving highly accurate 3D image even in low-power environments.

4. The sentence given from line 233-234 is not complete. The complete sentence is -

We used the microsphere (CPS532, SPI 233 Supplies), their refractive indices for the wavelength (532nm), and their diameters are given in Tabe 1.

Similarly, the sentence from lines 239-240 is incomplete. Complete sentence should be -

The refractive indices of the surrounding medium of microsphere and RBCs are shown in Table 1.

After implementing above mentioned minor revisions/corrections, the paper can be accepted for publication.

Comments on the Quality of English Language

There are few incomplete sentences in the manuscript as I have mentioned in the comments section. Please make corrections in those sentences.

Author Response

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