Monte Carlo Simulation of Electron Interactions in an MeV-STEM for Thick Frozen Biological Sample Imaging

Round 1

Reviewer 1 Report

MeV-STEM is becoming more and more important for the material science and thicker frozen biological sample Imaging. The authors have done great simulation by Monte- Carlo simulation. The results are interesting and could be used for the reference of instrument developing. However the document need to be greatly modified. Some problems couldn’t be ignored.

1.        Line 102-107 , Line 113-120 Line 123, From your manuscript, ‘where S_el is the elastic scattering cross-section, W is the solid angle,…., q is the scattering angle, W should be W, q₀ is the characteristic scattering angle below which 50% of the electrons are scattered into, l is the electron wavelength.’, q should be q, q₀ should be q₀, l should be λ. S_el should be σ_el, S_inel should be σ_inel , q_e should be q_e

2.        Line 165-172, all the symbols q should be replaced by q, dq should be replaced by dq, if it is solid angle, should be replace by dW. The same modifications should be done for  the manuscript.

3.        Normally we ask the authors to do data format in the form of scientific counts such as 1.7×10^-5, for the publication, 1.7E-05 is not allowed data format, although it is widely used in the output data format in computer simulation. So I suggest the authors to modify all the text in such format not only in the text also in the table 1.

4.        We need the coordinates name and units in the figure1, 2,3,4 like what you did in the figure 5.

5.        There are some arguments about your figure captions in the Figure 1-4 (also the explanation in the text), the description of ‘longitudinal beam profiles’ is not true. We call it transverse beam profile (actual ‘transverse beam envelope’), which it may be more accurate with the different projection depth (longitudinal). Would you like to do the modifications in the suggested way?

6.        Your abstract need to be rewritten, it should be more put in the introduction. You should give the emphasis on your results you described in the section 3. The content you mentioned in the abstract we didn’t see it from your manuscript.

MeV-STEM is becoming more and more important for the material science and thicker frozen biological sample Imaging. The authors have done great simulation by Monte- Carlo simulation. The results are interesting and could be used for the reference of instrument developing. However the document need to be greatly modified. Some problems couldn’t be ignored.

1.        Line 102-107 , Line 113-120 Line 123, From your manuscript, ‘where S_el is the elastic scattering cross-section, W is the solid angle,…., q is the scattering angle, W should be W, q₀ is the characteristic scattering angle below which 50% of the electrons are scattered into, l is the electron wavelength.’, q should be q, q₀ should be q₀, l should be λ. S_el should be σ_el, S_inel should be σ_inel , q_e should be q_e

2.        Line 165-172, all the symbols q should be replaced by q, dq should be replaced by dq, if it is solid angle, should be replace by dW. The same modifications should be done for  the manuscript.

3.        Normally we ask the authors to do data format in the form of scientific counts such as 1.7×10^-5, for the publication, 1.7E-05 is not allowed data format, although it is widely used in the output data format in computer simulation. So I suggest the authors to modify all the text in such format not only in the text also in the table 1.

4.        We need the coordinates name and units in the figure1, 2,3,4 like what you did in the figure 5.

5.        There are some arguments about your figure captions in the Figure 1-4 (also the explanation in the text), the description of ‘longitudinal beam profiles’ is not true. We call it transverse beam profile (actual ‘transverse beam envelope’), which it may be more accurate with the different projection depth (longitudinal). Would you like to do the modifications in the suggested way?

6.        Your abstract need to be rewritten, it should be more put in the introduction. You should give the emphasis on your results you described in the section 3. The content you mentioned in the abstract we didn’t see it from your manuscript.

Author Response

Dear Reviewer,

We appreciate the time and effort that you dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper.

We have incorporated the suggestions and modified the manuscript accordingly. Those changes are tracked by MS WORD within the manuscript.

Please see below, in blue, for a point-by-point response to your comments and concerns.

Best regards,

Liguo Wang

Reviewer 1:                             

MeV-STEM is becoming more and more important for the material science and thicker frozen biological sample Imaging. The authors have done great simulation by Monte- Carlo simulation. The results are interesting and could be used for the reference of instrument developing. However the document need to be greatly modified. Some problems couldn’t be ignored.

1. Line 102-107 , Line 113-120 Line 123, From your manuscript, ‘where S_el is the elastic scattering cross-section, W is the solid angle,…., q is the scattering angle, W should be W, q₀ is the characteristic scattering angle below which 50% of the electrons are scattered into, l is the electron wavelength.’, q should be q, q₀ should be q₀ , l should be λ. S_el should be σ_el , S_inel should be σ_inel , q_e should be q_e

Author response: we fixed all those symbol errors.

2. Line 165-172, all the symbols q should be replaced by q, dq should be replaced by dq, if it is solid angle, should be replace by dW. The same modifications should be done for the manuscript.

Author response: We fixed all those symbol errors.

3. Normally we ask the authors to do data format in the form of scientific counts such as 1.7×10-5 , for the publication, 1.7E-05 is not allowed data format, although it is widely used in the output data format in computer simulation. So I suggest the authors to modify all the text in such format not only in the text also in the table 1.

Author response: We changed the format.

4. We need the coordinates name and units in the figure1, 2,3,4 like what you did in the figure 5.

Author response: Coordinates name and units were added to figure 1-4.

5. There are some arguments about your figure captions in the Figure 1-4 (also the explanation in the text), the description of ‘longitudinal beam profiles’ is not true. We call it transverse beam profile (actual ‘transverse beam envelope’), which it may be more accurate with the different projection depth (longitudinal). Would you like to do the modifications in the suggested way?

Author response: We changed ‘longitudinal’ to ‘transverse’.

6. Your abstract need to be rewritten, it should be more put in the introduction. You should give the emphasis on your results you described in the section 3. The content you mentioned in the abstract we didn’t see it from your manuscript.

Author response: We rewrote the abstract.

Reviewer 2 Report

The study is interesting. But I miss the lack of experiments and comparison with them.

Is there any need to cite already well known equations.

The discussion needs to be supported by real experiments.

How close are these simuations to the real biological situation?

Author Response

Dear Reviewer,

We appreciate the time and effort that you dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper.

We have incorporated the suggestions and modified the manuscript accordingly. Those changes are tracked by MS WORD within the manuscript.

Please see below, in blue, for a point-by-point response to your comments and concerns.

Best regards,

Liguo Wang

Reviewer 2:

The study is interesting. But I miss the lack of experiments and comparison with them. Is there any need to cite already well known equations. The discussion needs to be supported by real experiments. How close are these simuations to the real biological situation?

Author response: Experiments conducted by Wolf et al. (references 10 and 11) utilized a STEM operating at 200 keV energy to image frozen biological samples, thereby increasing the imageable specimen thickness from 300-500 nm to 1,000 nm. Regarding the beam profile, while there are no experimental results available to our knowledge, the Wolf group performed computer simulations, yielding a beam radius close to what we observed (~2.7 nm) (reference 10). We cited these results in Section 3.2.

Reviewer 3 Report

In the peer-reviewed article, the authors investigate the topical problem of the interaction of 2 MeV electrons with thick frozen biological samples in scanning transmission or electron microscopes. For their analysis, the Monte Carlo method is used, which allows determining the distribution of electrons and the signals they cause on the detector. Thus, it is possible to obtain images of thick samples with a nanometer resolution.

Remark:

1. Table 1 shows the electron scattering cross section for H2O with only oxygen from 100 keV to 3 MeV. For a more effective analysis, it is necessary to expand the research and build appropriate graphical dependencies.

2. In Figure 6, it is advisable to consider more values of the studied sample depths (not only 5 μm and 10 μm). It may be worth plotting an appropriate calibration curve based on the results obtained. I think it may have some value in diagnostics, such as examining diseased or healthy tissue; if ill, what degree (1-mild, 2-moderate, 3-severe). Further studies may use machine learning techniques and ROC analysis to evaluate their sensitivity, specificity, and accuracy.

Author Response

Dear Reviewer,

We appreciate the time and effort that you dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper.

We have incorporated the suggestions and modified the manuscript accordingly. Those changes are tracked by MS WORD within the manuscript.

Please see below, in blue, for a point-by-point response to your comments and concerns.

Best regards,

Liguo Wang

Reviewer 3:

In the peer-reviewed article, the authors investigate the topical problem of the interaction of 2 MeV electrons with thick frozen biological samples in scanning transmission or electron microscopes. For their analysis, the Monte Carlo method is used, which allows determining the distribution of electrons and the signals they cause on the detector. Thus, it is possible to obtain images of thick samples with a nanometer resolution.

Remark:

1. Table 1 shows the electron scattering cross section for H2O with only oxygen from 100 keV to 3 MeV. For a more effective analysis, it is necessary to expand the research and build appropriate graphical dependencies.

Author response: We plot it as a figure 1 in the top of page 4.

2. In Figure 6, it is advisable to consider more values of the studied sample depths (not only 5 μm and 10 μm). It may be worth plotting an appropriate calibration curve based on the results obtained. I think it may have some value in diagnostics, such as examining diseased or healthy tissue; if ill, what degree (1-mild, 2- moderate, 3-severe). Further studies may use machine learning techniques and ROC analysis to evaluate their sensitivity, specificity, and accuracy.

Author response: While it is possible to consider additional values, it would result in a linear increase in the number of figures required. In order to conserve space, we opted to focus on sample depths of 5 μm and 10 μm. The former demonstrates the increase in beam size at the top surface, with the beam size remaining almost constant in the top 1-2 μm region. The latter illustrates a wider beam profile from 3 μm to 6 μm, with minimal change thereafter.

Round 2

Reviewer 1 Report

MeV-STEM is becoming more and more important for the material science and thicker frozen biological sample Imaging. The authors have done great simulations by Monte- Carlo simulation. The results are interesting and could be used for the reference of instrument developing. The authors have done the great modifications according to the review comments one by one. The reviewer thanks to the authors.

 The figure 1 has been added. The figure caption still has a small typing error of elastic scattering (q_e), not q0. I agree all the modifications done by the authors. It looks much better than before. I suggest the editor to accept this paper to publish in our magazine.