A Three-Dimensional Tracking Method with the Self-Calibration Functions of Coaxiality and Magnification for Single Fluorescent Nanoparticles
Round 1
Reviewer 1 Report
This manuscript, “Single fluorescent nanoparticles three-dimensional tracking method with self-calibration functions of coaxiality and magnification,” by Mao et al. provides a detailed analysis and proof of functionality of an imaging system for 3D single-particle tracking. They incorporate a cylindrical lens to correlate the point spread function shape the particle location along the z-axis. They analyze how the design of the imaging system affects the accuracy of the particle’s location measurement and its effectiveness in performing 3D particle localization. The demonstrate their theoretical approach experimentally and perform 3D single-particle tracking on nanoparticles of varying diameter with varying magnification.
The broad idea of using a cylindrical lens to reveal a particle’s 3D location is, as the authors state, not new. Broadly, it is unclear what this manuscript provides that is not previously discussed in greater length in prior publications. Their use of optical transfer matrices to analyze the rays passing through their system without rotational symmetry is appreciated while making the paraxial approximation. Their single-particle images clearly demonstrate elliptical point spread functions that correlate with particle Z location.
However, there are numerous issues with this manuscript that limit this reader’s enthusiasm. Namely, the manuscript does not adequately describe the assumptions made in the theoretical approaches or the limits of the theory in describing the experimental results. The figures contain multiple shortcomings that are inaccurate (e.g., units) or reduce clarity (e.g., 3D graphs that would be clearer in 2D). Finally, a major point of the manuscript is that the imaging system provides accurate MSD analysis. However, no discussion is given to how the acquired MSD results are known to be correct. The manuscript must better describe what the provided MSD results reveal about the imaging system.
The display used in Fig 3 does not allow the reader to see that the X and Y axis width ratio does not change with Delta. A simple top view of this 3D plot would help such that the lines of constant X and Y axis width ratio would appear perpendicular to the Delta axis.
The claim of “the nanoparticle image intensity distribution is constant for an object plane” may be consistent with the mathematical approximations used, it is clearly not experimentally valid in the presence of a camera with dark counts and imperfect sensitivity. The manuscript must better describe the mathematical approximations used and their limitations.
This phrase on line 110 is not clear as written. Please reword “the focal position for one-point light source is not existed.”
In Fig 4 and 5, it seems like the effects on the Measurement Error should change with d0 and d1 with a sensitivity difference scaled by n_i = 1.33. Such that an epsilon change in d0 should be equivalent to an epsilon/n_i change in d1. However, this does not seem to be the case in this figure. Please explain.
The y-axis labels in Figs. 4, 5,6, and 8, for example, should include the units μm not “um.”
Typical optical systems and applications of transfer matrices use spherical lenses with the lens size significantly smaller than the radius of curvature of the lens. This allows the lenses to be more accurately modeled in the paraxial approximation. However, the schematics of this figure show the spherical lenses to be spheres. This would imply the paraxial approximation and the transfer matrix L1 is far from accurate. Please explain.
The tilted photograph in Fig. 7 makes it difficult to compare to the schematic. It is unclear which parts of the photograph correspond to which parts of the schematic.
The line spacing on lines 184-193 is not consistent with the rest of the text.
It is unclear what the differences are between the first, second, and third MSD in Fig. 10.
Author Response
Please see the attachment,thanks.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this paper, the authors provided a simple method to improve the real-time 3D particle tracking. They used cylindrical lens, and a spherical lens was placed between the cylindrical and cover glass in order to self-calibrate coaxiality and magnification. The calibrated functions hold for the various 3D tracking.
Unfortunately, there is nothing very novel in their methods and the authors failed to be more ethical in referencing. There are absolutely a bunch of the similar 3D detection schemes and now the novel nano-probe systems may be worth referenced.
So I suggest this paper be accepted after referring to a few more previous studies which dealt with (e.g. upconverting nanoparticles) tracking in 3D. The additional minor suggestions are as follows.
In Fig 1, Cylindrial --> Cylindrical In Fig 10 (legend), are first, second, and third correspond to 50 nm, 100 nm, and 200 nm, respectively? Or vice versa?
Comments for author File: Comments.docx
Author Response
Please see the attachment,thanks.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The revised manuscript is significantly improved and no significant errors were noted.
