Characterization and Optimization of the Biplane Distance in Three-Dimensional Single-Molecule Localization Microscopy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript addresses a highly practical and important issue in 3D-SMLM, with clear research logic, rigorous theoretical derivation, and well-designed experiments. The theoretical simulation and experimental results are in good agreement, supporting convincing conclusions. The manuscript is suitable for publication after minor revisions addressing the following comments.

1. The tunable biplane detection module developed in this work provides valuable guidance for constructing high-performance 3D-SMLM systems. However, the current module uses a relatively complex and high-cost design with ultra-high-precision piezoelectric actuators. Given the experimental results shown in the manuscript, such high precision may not be necessary for the reported measurements. I suggest the authors add a discussion to evaluate whether the biplane adjustment module can be simplified for more cost-effective applications.
2. The authors state that their calculation results under small aberrations (Fig. 4) are similar to the ideal aberration-free case, and that PSF optimization was applied in experiments to suppress large aberrations. Nevertheless, aberration-correction components such as a deformable mirror are not shown in the optical setup diagram (Fig. 1(a)). The authors should clearly explain how the PSF optimization method was specifically implemented.
3. The authors are encouraged to upload the calculation code as supplementary materials to provide a useful reference for other researchers in this field.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by X. Wang et al. presents a study on the axial localization accuracy of biplane single-molecule localization microscopy, both theoretically and experimentally. The manuscript contains in-depth discussion with sufficient details provided. The writing is also well-done. I have only a few minor comments.

1. Figure 3: The authors discussed the results extensively focusing on the values they obtained. However, this got me lost. Could the authors provide some physics intuition on why there is an ‘optimal’ value of biplane distance.
2. Line 210: To be technically rigorous, the system is not fully aberration-free. Spherical aberration caused by the planar interface (Figure 1b) shall be pointed out in the text. In addition, could the authors explain how they treated the spherical aberration theoretically (which may blur the focal plane)?

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Summary:
This manuscript investigates how biplane distance affects axial localization precision in biplane 3D single-molecule localization microscopy (3D-SMLM). The authors combine CRLB-based analysis with experimental validation using fluorescent beads and mitochondrial samples, and conclude that a biplane distance of 0.380 µm yields superior axial precision compared with 0.200 µm and 0.599 µm under their imaging conditions. The topic is relevant to the optimization of practical 3D-SMLM systems, and the work appears to provide both theoretical and experimental value.

General Assessment:
The study addresses an important technical parameter in biplane 3D-SMLM and presents a practically useful conclusion. The manuscript’s central claim—that 0.38 µm is the optimal biplane spacing for the stated setup and axial range—is clear and supported in the conclusion by both theoretical analysis and experiments. The work is potentially useful for researchers designing or tuning biplane detection modules.
Major comments：
1. While the authors correctly identify 0.38 μm as the optimum for their setup, this value is highly dependent on the Numerical Aperture and Wavelength. The report would be strengthened if the authors provided a dimensionless ratio or a simplified formula to help other researchers calculate the optimum for different NAs (e.g., oil vs. water immersion).

2. The current analysis is centered on an axial range of up to ±1.0 um. As mammalian cells typically have thicknesses of approximately 3–6 um, a short discussion of how the optimal biplane distance may change for larger imaging depths would improve the manuscript’s relevance and practical applicability.
Recommendation:
Minor revision.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf