Deformation Measurements of Neuronal Excitability Using Incoherent Holography Lattice Light-Sheet Microscopy (IHLLS)
Round 1
Reviewer 1 Report
Deformation measurements of neuronal excitability using incoherent holography lattice light-sheet microscope (IHLLS)
M. Potcoava, J. Art, S. Alford, C. Mann
The authors describe the recent technique of incoherent holography lattice light-sheet microscopy (IHLLS) which for measurements of neuronal size. The technique combines a self-interference holography system in the detection pathway of a lattice light-sheet microscope which allows 3D images to be acquired from a specimen without moving either the specimen or the objective lens. This yields more reliable reconstruction of 3D information and potentially faster imaging. The IHLLS technique is then applied to imaging neurons under different osmotic pressures by changing the medium surrounding them.
The manuscript is well written and quite clear, although I had a bit of difficulty getting my head around the different modalities and acronyms (IHLLS, IHLLS 1L, IHLLS 2L).
I think the subject of this manuscript is very suitable for the Holography Special Issue of Photonics but, in its current form, is not suitable for publication. Please see my comments below.
I am not sure what the focus of the manuscript is. The IHLLS technique has been reported recently (ref 21) and so I do not think this manuscript should just repeat that information. Instead, I think the focus should be on the application of this new technique for a study (deformation measurements of neuronal excitability). However, I feel the focus is very much on the description of the technology and very little space is given to discussing the deformation measurements.
Major comments:
- One concern regarding the manuscript is that this technique (IHLLS) has already been reported recently (ref 21) without modification (as far as I can tell). In this case, large sections of Section 3.1 (the bulk of the manuscript) are unnecessarily repeated. Panels c, e-h in Fig 2 match very closely with Fig 2 in ref 21. This figure could do with more attribution or really could just be removed and a citation given. Section 3.1 could certainly be made more concise, this would allow for more analysis of the neuronal images (See next point).
- Another large concern is that the title starts with "Deformation measurements..." and there doesn't seem to be a lot of measurement or analysis of the acquired images. The neurons are imaged in 2 different osmotic pressures (isotonic and hypo osmotic in Fig 4). The only discussion of these images is in lines 287 - 289: "It is clear from this that the soma and the dendrites all showed an increase in volume.
Minor comments:
- Line 150: Check "LLS (green) and IHLLS (red)".
- Line 268: New paragraph after "[Fig 3c]." would make it clearer that you are talking about a different modality (IHLLS 2L instead of IHLLS 1L).
- Line 277: The phases are given in radians but the symbol is for degrees.
- Line 298: "In a prior study..." - give the citation for this study.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript describes a lattice light-sheet microscopy system with incoherent digital holography. The optical system is adequately designed though ray optics and wave optics theories. The described theory behind the proposed system is correct, the reference list is appropriate, and the results are interesting. The presented work shows an attractive application of incoherent digital holography, and thus it would be useful in optics and biology fields. Therefore, in my opinion, the presented manuscript is suitable for publication.
However, I have one question. The authors multiplexed two diffractive lenses randomly to create a single phase pattern to be displayed on an SLM, as described in ref [26]. I think this approach causes diffused noise in reconstructed images. A polarization multiplexing method [27] by using the polarization property of the SLM would be able to reduce the noise. Why did the authors use the random multiplexing method [26] for the phase pattern? Is the effect of noise negligible?
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
The presentation is very poor and therefore the scientific findings cannot be judged.
Issues with presentation: some references are in bold font some are not, double spaces throughout the text, subscripts are not always subscripts (MgSO4 should be MgSO_4), missing full stops, using “^2” instead of superscript, etc. It seems that the authors did not check their manuscript before submission.
References must be provided to previous literature. For example, after the sentence “Lattice Light Sheet Microscopy provides a method for generation of 3D imaging using a self-reinforcing lattice of Bessel beams as an excitation source and imaging the subsequent fluorescence orthogonally”. The whole sections need a figure with the illustration of the principle.
The terminology must be corrected throughout the text. For example “a plane wave light emitted from an infinity corrected objective” makes no sense because objectives do not emit light. Long sentences must be avoided, for example “The SLM has multiple roles here: to split a parallel beam of light in two spherical beams, to create two different diffractive lenses on the same SLM with randomly distributed pixels by lens multiplexing [26, 29], to phase-shift to change the phase on one of the diffractive lenses, and phase modulation through adjustment of the spatial distribution of the incoming light such as the phase, polarization state, and intensity of the incoming beam in order to get the maximum phase modulation possible” – not clear at all.
Fig. 1(a) can be removed, it is not clear what is happening in this photo.
Fig. 2: it is not ok to use screen-shots of some technical drawings as figures. The resolution in these images is very low, one cannot see the text and symbols in these figures, so these images only confuse the reader.
“520 center wavelength, 40 nm band pass filter BPF” – very unclear and unconventional writing, does 520 has some units? nm?
From line 210: the coordinate system is not introduced and essential to understand the principle. What are (x,y) and (u,v) coordinates? Are they in the same or different planes? A drawing would be helpful.
Scalebars must be provided in all images.
Why in Fig. 4(e)-(i) the units of phase are microns?
The Authors should respect the rules of scientific writing (International Organization for Standardization (ISO) International Standard ISO 31-0:1992 to ISO 31-13:1992). A brief sum-up of the rules:
- physical values should be written in italic
- units, subscripts and text should be written in non-italic
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Thank you for your revisions to the manuscript.
The additions to the abstract (lines 15-18) and introduction (lines 82-86) have helped to define the focus of the manuscript and should aid the reader's understanding of the different (but similar) acronyms used.
The IHLLS-2L technique has been covered in ref 24 with an identical microscope (as far as I can tell - the focal lengths stated are identical) and I still think re-reporting this amount of information is unnecessary if there have been no changes from the previous description.
The additions to section 3.2 (lines 298-308) give a much better quantitative analysis of the results and I like the corresponding annotations to Fig 4.
Author Response
Point-by-point response in red
Reviewer 1
Author Response: We thank the reviewer for the comments. The remaining question is expressed in his question as follows:
Reviewer Comment: The IHLLS-2L technique has been covered in ref 24 with an identical microscope (as far as I can tell - the focal lengths stated are identical) and I still think re-reporting this amount of information is unnecessary if there have been no changes from the previous description.
Author Response: We understand the reviewer’s question on this point, but we respectfully request to keep it as is stands because we feel that when the manuscript is written without this explanation it is very burdensome to refer for key parts back to the earlier manuscript.
Author Response File: Author Response.pdf
Reviewer 3 Report
Unfortunately, hardly anything was corrected and the manuscript is still very negligibly prepared. The scientific writing rules are still completely ignored. Although this is quite easy to correct such things, no corrections have been made. A few (but not all) examples:
Line 162: “band pass 162 filter BPF with bandwidth ∇?=40??;” – a nabla operator?
Line 214: ” … lenses, fSLM,, fd1, and fd2, uploaded… “ – even punctuation here was not corrected
Line 217: “Q(b) = exp[iπb?−1(x2+?2)]”
Line 260: “????= 400 mm”
Line 305: “the x-y plane”
Line 315: “beams (FOV 208 μm2)”
Eq. (3) It should be “+ 2∗?1(?,?)?2(?,?)cos (?1(?,?)−?2(?,?)−?) ”
Eq. (4) Why the argument of function is written as subscript
Eq. (5) Why there is a zero on the left side?
Fig. 3. It is not explained what are those image: intensity, amplitude or phase?
Fig. 4 The Bessel beams are displayed in the upper left corner of each xy-projection to show the orientation of the beams – There are no Bessel beams in upper left corners, there are only letters labelling the panels.
Author Response
Point-by-point response in red
Reviewer 3
Author Response: We would like to thank the reviewer for his/her care and patience in this review. We have attempted to carefully edit the document for errors in addition to responding to the individual highlighted comments below.
Reviewer Comment: Line 162: “band pass 162 filter BPF with bandwidth ∇?=40??;” – a nabla operator?
Author Response: This is corrected to read Dl=40nm
Reviewer Comment: Line 214: ” … lenses, fSLM,, fd1, and fd2, uploaded… “ – even punctuation here was not corrected
Author Response: The excess commas were removed
Reviewer Comment: Line 217: “Q(b) = exp[iπb?−1(x2+?2)]”
Author Response: We are unsure of this comment but (b) in Q(b) refers to -1/f in the preceding equation
Reviewer Comment: Line 260: “????= 400 mm”
Author Response: SLM italics are removed.
We have changed subscripts to non-italics, and we defer to the editor for the correct representation.
Reviewer Comment: Line 305: “the x-y plane”
Author Response: corrected to xy plane
Reviewer Comment: Line 315: “beams (FOV 208 μm2)”
Author Response: This is corrected throughout to 208 x 208 μm2
Reviewer Comment: Eq. (3) It should be “+ 2∗?1(?,?)?2(?,?)cos (?1(?,?)−?2(?,?)−?) ”
Author Response: corrected
Reviewer Comment: Eq. (4) Why the argument of function is written as subscript
Author Response: corrected
Reviewer Comment: Eq. (5) Why there is a zero on the left side?
Author Response: This is a phi but the font was odd. This is corrected
Reviewer Comment: Fig. 3. It is not explained what are those image: intensity, amplitude or phase?
Author Response: This is corrected. ‘The intensity images using the IHLLS 1L mode were recorded only for the diffractive lens with phase shift ’
Reviewer Comment: Fig. 4 The Bessel beams are displayed in the upper left corner of each xy-projection to show the orientation of the beams – There are no Bessel beams in upper left corners, there are only letters labelling the panels.
Author Response: Relabeled to say:
The Bessel beams are displayed in the right-hand gray panel of each xy-projection – this is corrected in the legend.
Author Response File: Author Response.pdf
