Focusing of Microcrystals and Liquid Condensates in Acoustofluidics
, Iwona Ziemecka 1, Peter Tompa 2, Wim De Malsche 1 and Dominique Maes 2,*Round 1
Reviewer 1 Report
The manuscript entitled “Manipulation of crystals and high-density droplets of proteins inacoustofluidics” by Gelin et al. is sound and of high interest for the community. The experimental work is throughout most well and detailed executed and described.
The manuscript certainly deserves publication.
The authors describe an interesting method and approach to manipulate crystals as well as liquid dense clusters in microfludic compartments via acoustic radiation.
Important today as serial crystallography is established at high intensive synchrotron radiation sources and Free-Electron Laser radiation sources, and particle imaging is coming up as well at FELs. Both methods utilize either on chip, tape drive or jet technologies to deliver sample suspensions to the beam. For the last the development of manipulation techniques to optimize the sampe consumption, improving the hit rates are this days under continious development. Therefore the topic of this manuscript is timely, considering also other applications in bioanalytical or pharmaceutical production and scoring processes.
The authors should try to highlight this aspects as possible.
Some further minor points should be considered as well:
The term “high-density droplets”, which is indeed relevant in the field may be changed to a more common one, e.g. “clusters” instead of “droplets” for clarity (also in the keyword list).
In the introduction the authors mention and reference eukaryotic cells, the may include and consider also latest references as: Elio A. Abbondanzieri· Anne S. Meyer, 2018, Current Genetics, doi.org/10.1007/s00294-018-00927-x, Steven Boeynaems et al., June 2018, Vol. 28, No. 6
Why the authors have choosen the frequency generator mentioned lines 83-84 ?
Confusing, line 104 the term “dense liquid protein clusters” is used, however are some of these clusters in the lysozyme solution (before adding an additional precipitant), which need to be removed, as mentioned in line 104?
The text below figure 2 “focusing of lysozyme” should be changed to “focusing of lysozyme microcrystals”
Line 179 is mentioning droplet fusion should be studied further. Was there already an observation with the setup (figure 3) that the droplets fuse as a consequence of focusing and centering ?
The authors should add information about the size of hnRNPA2 and the size distribution of the “small” and “large” high density clusters in the experiment (figure 3).
1-2 more sentences about the potential or future setup/instrument for size-based particle separation should be considered.
Further references on the use of acoustic waves to manipulate crystals and biological macromolecular assemblies, which are rather diverse (e.g. for sample delivery, sorting and increasing the number of hits in a crystallization screen) should be included. Likewise, to emphasize more applications, as mentioned before in areas where particle sorting is this days of highly relevant, e.g. serial crystallography and single particle imgaing.
Author Response
We are grateful for the valuable comments of the reviewer, which were considered in the revised version of our paper as described hereunder. For the sake of clarity, reviewers’ comments are in black while our answers are in blue.
Comments:
The manuscript entitled “Manipulation of crystals and high-density droplets of proteins in acoustofluidics” by Gelin et al. is sound and of high interest for the community. The experimental work is throughout most well and detailed executed and described.
The manuscript certainly deserves publication.
The authors describe an interesting method and approach to manipulate crystals as well as liquid dense clusters in microfluidic compartments via acoustic radiation.
Important today as serial crystallography is established at highly intensive synchrotron radiation sources and Free-Electron Laser radiation sources, and particle imaging is coming up as well at FELs. Both methods utilize either on chip, tape drive or jet technologies to deliver sample suspensions to the beam. For the last the development of manipulation techniques to optimize the sample consumption, improving the hit rates are this day under continuous development. Therefore, the topic of this manuscript is timely, considering also other applications in bioanalytical or pharmaceutical production and scoring processes.
1. The authors should try to highlight this aspect as possible.
We agree with both comments. The introduction and abstract have substantially been adapted to put more emphasis on the FEL application of our technology (page no 1-2).
In the results and discussion the following line has also been added to the manuscript (line 153-155):
This is especially of relevance in current serial crystallography experiments were a thin jet of crystals is brought in the X-ray beam.
Some further minor points should be considered as well:
2. The term “high-density droplets”, which is indeed relevant in the field may be changed to a more common one, e.g. “clusters” instead of “droplets” for clarity (also in the keyword list).
We agree with this comment. In the title the term “high density droplets of proteins” has been changed to liquid condensates. This has also been done in the abstract (line 12 and 15) and in the main text (line 51, 55, 65 and 190).
3. In the introduction the authors mention and reference eukaryotic cells, the may include and consider also latest references as: Elio A. Abbondanzieri· Anne S. Meyer, 2018, Current Genetics, doi.org/10.1007/s00294-018-00927-x, Steven Boeynaems et al., June 2018, Vol. 28, No. 6
Both references are mentioned in the paper (lines 50-51).
4. Why the authors have chosen the frequency generator mentioned lines 83-84?
The frequency generator covers a broad spectrum of waveforms and frequencies. Moreover, it allows to generate an arbitrary wave and has two channels offering a high flexibility.
5. Confusing, line 104 the term “dense liquid protein clusters” is used, however are some of these clusters in the lysozyme solution (before adding an additional precipitant), which need to be removed, as mentioned in line 104?
We agree that this is confusing, the sentence has been changed to (line 101):
Before experiments, the lysozyme solution was filtered with a 0.1µm PTFE syringe filter to remove impurities.
6. The text below figure 2 “focusing of lysozyme” should be changed to “focusing of lysozyme microcrystals”
This has been adapted in the manuscript (line 155, 166, 169).
7. Line 179 is mentioning droplet fusion should be studied further. Was there already an observation with the setup (figure 3) that the droplets fuse as a consequence of focusing and centering?
Droplet fusion was indeed already observed during some of the experiments. However, investigating this in detail is beyond the scope of the current contribution. Droplet fusion will be further investigated and published in the future.
8. The authors should add information about the size of hnRNPA2 and the size distribution of the “small” and “large” high density clusters in the experiment (figure 3).
hnRNPA2 is a disordered and very dynamic protein which changes significantly its conformation over time. Therefore, we can roughly estimate its size based on the number of amino acids. The additional information was included in line 105-107.
The small droplets refer to droplets formed at the beginning of the experiment while the large droplets are droplets obtained after 1h. Relevant description in the manuscript was included in lines: 193-195.
9. 1-2 more sentences about the potential or future setup/instrument for size-based particle separation should be considered.
The following sentences have been added to the manuscript (lines 228-233):
In a typical set up, microcrystals of different sizes would be inserted in the microfluidic channel at the outside edges, separated by an inert stream in the center (see Figure 1.b). Upon activation of the acoustic field and with the correct process parameters chosen (flow velocity, voltage, number of outlets, etc.), the largest crystals focus rapidly in the center of the channel, where they can be extracted. The smaller crystals, however, have not focused yet and can be collected at different outlets.
10. Further references on the use of acoustic waves to manipulate crystals and biological macromolecular assemblies, which are rather diverse (e.g. for sample delivery, sorting and increasing the number of hits in a crystallization screen) should be included. Likewise, to emphasize more applications, as mentioned before in areas where particle sorting is this day of highly relevant, e.g. serial crystallography and single particle imaging.
The introduction and abstract have been substantially adapted to put more emphasis on the FEL application of our technology (page 1-2).
Reviewer 2 Report
The authors present a proof of concept for a microfluidic platform in which they would like to separate crystals and droplets using acoustofluidics. The manuscript is well written and clear. They show results the for miconazole crystals and high density droplets. The authors also show a simulation using polystyrene beads of different sizes.
What I felt could significantly improve their manuscript is some statistics on these two experiments. I would like to see size distribution etc. Another experiment that may improve the manuscript a lot is to actually take the polystyrene beads and do the experiment. A good correlation with their simulation will strengthen the value of this platform significantly.
Author Response
We are grateful for the valuable comments of the reviewer, which were considered in the revised version of our paper as described hereunder. For the sake of clarity, reviewers’ comments are in black while our answers are in blue.
The authors present a proof of concept for a microfluidic platform in which they would like to separate crystals and droplets using acoustofluidics. The manuscript is well written and clear. They show results the for miconazole crystals and high density droplets. The authors also show a simulation using polystyrene beads of different sizes.
What I felt could significantly improve their manuscript is some statistics on these two experiments. I would like to see size distribution etc.
Size and standard deviation of crystals and droplets were added to the manuscript (line 156, 170, 192). Experimentally obtained migration velocities were fitted with the simulations in a different paper (under revision). Herein we obtained a satisfactory standard deviation.
Another experiment that may improve the manuscript a lot is to actually take the polystyrene beads and do the experiment. A good correlation with their simulation will strengthen the value of this platform significantly.
We have recently developed a 3D particle image velocimetry instrument with which we will provide detailed 3D velocity vectors of fluorescent polystyrene particles. We will treat this is in a follow-up publication.
Reviewer 3 Report
Overall impression: The authors describe a technique for manipulating the location of dense objects within a microfluidic stream, including protein crystals, pharmaceutical crystals, or dense liquid condensates. The results of great significance to applications such as manipulating the location of protein crystals within a moving XFEL injector stream. The results are innovative and relevant to many applications in structural biology, pharmacology, cell biology, and drug discovery. Overall, the manuscript is powerful and potentially paradigm shifting. However, there are problems with clarity and completeness:
· The title and abstract are overly technical and will likely go un-noticed by many groups that could potentially benefit from this application. In particular, the XFEL and serial crystallography communities are briefly mentioned in the introduction, and then completely forgotten thereafter.
· The introduction is missing key opportunities to reach out to these and other communities, and also fails to mention any prior applications of acoustic methods to manipulation of crystal samples or to combining pharmaceutical chemicals with protein crystals. In the absence of these important citations, the reader misses the key innovation demonstrated in this manuscript, which has eluded many groups that previously worked with acoustic crystal manipulation, namely, the ability to manipulate dense objects within moving streams.
· The methods and results sections of the manuscript are well written and clear, but could benefit from proofreading for minor grammatical errors.
· Like the introduction, the conclusion misses the opportunity to contextualize the capability demonstrated here with the needs of serial crystallography and FEL communities.
Specific comments:
Title: This technique will likely be of broad utility to the XFEL community and to the serial crystallography community, but neither community is likely to understand the title. “High-density droplets of proteins” are technical terms that will not be recognized by groups that could derive great benefit from acoustic sample manipulation techniques such as this. It is probably not possible to change the title at this time, but this should be clarified as early in the manuscript as possible (for example in the beginning of the abstract).
Abstract: “high-density droplets” is not common terminology in structural biology. Crystallographers know that protein (and protein crystals) are significantly denser than water. It would be better to specify “high density materials such as crystals or liquid condensates.”
Keywords: Keywords of interest to developers are included, but not keywords of interest to potential users. Suggested keywords that will be of interest to potential users of this technique: (i) “serial crystallography”, (ii) “free electron laser”, (iii) “micro crystals”, (iv) “crystal harvesting”.
Introduction
Line 27: “Microfluidic technology offers exceptional control over the crystallization conditions, making it a very interesting tool to investigate crystallization processes.” Confusing terminology, crystallization processes are not themselves being investigated by microfluidic technology. Rather, microfluidic technology is a tool used to investigate a biological problem. This sentence should be replaced with clearer language. An example is “Microfluidic technology offers exceptional control over crystallographic investigations of biological problems, and microfluidic manipulation of experimental building blocks are a powerful tool for crystallographers.”
Line 33: “Many different devices such as optical tweezers [5], surface acoustic waves [4] and robotic devices [6], [7] have been made to automate and manipulate crystals.” Sound waves have also been used to manipulate crystals, and some (or all) of these citations should be in the manuscript:
· Sound waves used to arrange crystals in an ordered array:
o Guo, F., Zhou, W., Li, P., Mao, Z., Yennawar, N.H., French, J.B. and Huang, T.J., 2015. Precise manipulation and patterning of protein crystals for macromolecular crystallography using surface acoustic waves. small, 11(23), pp.2733-2737.
· Sound waves used to harvest protein crystals for data collection in synchrotrons:
o Cuttitta, C.M., Ericson, D.L., Scalia, A., Roessler, C.G., Teplitsky, E., Joshi, K., Campos, O., Agarwal, R., Allaire, M., Orville, A.M. and Sweet, R.M., 2015. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening. Acta Crystallographica Section D: Biological Crystallography, 71(1), pp.94-103.
· Sound waves used to deliver protein crystals to a FEL:
o Roessler, C.G., Agarwal, R., Allaire, M., Alonso-Mori, R., Andi, B., Bachega, J.F., Bommer, M., Brewster, A.S., Browne, M.C., Chatterjee, R. and Cho, E., 2016. Acoustic injectors for drop-on-demand serial femtosecond crystallography. Structure, 24(4), pp.631-640.
In terms of acoustic manipulation, the key innovation demonstrated in this manuscript is the first demonstration of manipulation of moving protein crystals. All other methods used to date have manipulated protein crystals that were initially at rest (sound waves were then used to impart momentum to the crystals, which then put them into motion). Given the enormous importance of jet technology to FEL crystal injection, the inability to acoustically manipulate moving crystals has been a crippling limitation in acoustic implementation at XFEL sources. This manuscript describes a highly important and likely paradigm shifting innovation, but this innovation has not taken place in a vacuum. Readers will be interested in the history of acoustic crystal manipulation, which will help put into context the key innovation demonstrated here: the manipulation of moving crystals, such as crystals within a FEL jet.
Methods/Results:
Line 82: Grammar, use “are” instead of “is”: “A schematic representation and a picture of the 82 acoustofluidic chip is are shown …”
Line 94: Grammar, use “were” instead of “was”: “A broad spectrum of biological compounds was were introduced …”
Line 101/105/122: Please be consistent in usage of chemical symbols or chemical names. If you are going to say “sodium acetate”, please also say “sodium chloride”. Note that it is fine to use acronyms for very long buffer names (eg MES, line 129).
Line 130: Readers who work on FEL problems will be very interested in the fluid velocity, rather than the volumetric flow rate.
Line 153: Crystal fowling is a HUGE problem in FEL injectors also. For some proteins, it is typical for only a few minutes of data to be obtained before the injector must be removed and cleaned, which can take as much as one hour to accomplish (the CXI station now features an automatic cleaning cycle, but it rarely works). This would be a good place to mention this huge advantage of combining the method described in this manuscript with FEL protein crystal injection, in preparation for a more thorough treatment in the discussion.
Conclusion:
Line 212: “Increasing the local density of crystals is of great relevance for the current crystallography methods.” The sample utilization rate of XFEL injectors is very poor. High flow rate crystal injection is “thin”, such that the XFEL beam profile covers a significant portion of the crystal stream … but high flow rate injection causes the overwhelming majority of the crystals to be missed because they traverse the beam path between XFEL pulses (typical sample utilization rates are much less than 1%, and sometimes closer to 1 part per million). To avoid this problem, the sample injection must be greatly slowed using viscous media, for example using LCP, PEO, or agarose. However, viscous media cause the crystal injection to become very “thick”, usually around 100 um or more. For a 100 um crystal stream, a 1 um beam will miss 99% of the sample, hence again leading to very low sample utilization rates. If BAW methods can be used to arrange crystal samples within an FEL flowing injection stream, then please mention the remarkable potential savings in terms of sample utilization. Sample utilization is currently one of the biggest problems with the FEL solution to the structural biology problem. If you have a solution to this conundrum, please discuss it here. If not, it would be good to mention why not (for example, maybe it is not possible to arrange protein crystals within viscous media.
Comments for author File: 
Comments.pdf
Author Response
We are grateful for the valuable comments of the reviewer, which were considered in the revised version of our paper as described hereunder. For the sake of clarity, reviewers’ comments are in black while our answers are in blue.
Overall impression: The authors describe a technique for manipulating the location of dense objects within a microfluidic stream, including protein crystals, pharmaceutical crystals, or dense liquid condensates. The results of great significance to applications such as manipulating the location of protein crystals within a moving XFEL injector stream. The results are innovative and relevant to many applications in structural biology, pharmacology, cell biology, and drug discovery. Overall, the manuscript is powerful and potentially paradigm shifting. However, there are problems with clarity and completeness:
· The title and abstract are overly technical and will likely go un-noticed by many groups that could potentially benefit from this application. In particular, the XFEL and serial crystallography communities are briefly mentioned in the introduction, and then completely forgotten thereafter.
· The introduction is missing key opportunities to reach out to these and other communities, and also fails to mention any prior applications of acoustic methods to manipulation of crystal samples or to combining pharmaceutical chemicals with protein crystals. In the absence of these important citations, the reader misses the key innovation demonstrated in this manuscript, which has eluded many groups that previously worked with acoustic crystal manipulation, namely, the ability to manipulate dense objects within moving streams.
· The methods and results sections of the manuscript are well written and clear, but could benefit from proofreading for minor grammatical errors.
· Like the introduction, the conclusion misses the opportunity to contextualize the capability demonstrated here with the needs of serial crystallography and FEL communities.
Specific comments:
1. Title: This technique will likely be of broad utility to the XFEL community and to the serial crystallography community, but neither community is likely to understand the title. “Highdensity droplets of proteins” are technical terms that will not be recognized by groups that could derive great benefit from acoustic sample manipulation techniques such as this. It is probably not possible to change the title at this time, but this should be clarified as early in the manuscript as possible (for example in the beginning of the abstract).
We agree with the comments. The title has been changed to (line 2):
Focusing of microcrystals and liquid condensates in acoustofluidics
2. Abstract: “high-density droplets” is not common terminology in structural biology. Crystallographers know that protein (and protein crystals) are significantly denser than water. It would be better to specify “high density materials such as crystals or liquid condensates.”
We agree that the abstract was overly technical for readers from another field. The abstract has been substantially changed.
3. Keywords: Keywords of interest to developers are included, but not keywords of interest to potential users. Suggested keywords that will be of interest to potential users of this technique: (i) “serial crystallography”, (ii) “free electron laser”, (iii) “micro crystals”, (iv) “crystal harvesting”.
We agree with this. The keywords have been changed to:
microcrystals, liquid-liquid phase separation, microfluidics, acoustic radiation, particle separation, focusing, serial crystallography, free electron laser, crystal harvesting.
4. Introduction
Line 27: “Microfluidic technology offers exceptional control over the crystallization conditions, making it a very interesting tool to investigate crystallization processes.” Confusing terminology, crystallization processes are not themselves being investigated by microfluidic technology. Rather, microfluidic technology is a tool used to investigate a biological problem. This sentence should be replaced with clearer language. An example is “Microfluidic technology offers exceptional control over crystallographic investigations of biological problems, and microfluidic manipulation of experimental building blocks are a powerful tool for crystallographers.”
This has been changed to the following text (line 26-30):
‘Microfluidic technologies benefit from a high surface to volume ratio, well-defined residence time and precise control over mass and heat transfer. This allows for optimization of crystallization conditions and the formation of high-quality crystals [1]–[3]. Moreover, microfluidic technology offers exceptional control for crystallographic investigations or other biological phenomena. Finally, microfluidics allows to precisely manipulate material inside the microfluidic device.’
Line 33: “Many different devices such as optical tweezers [5], surface acoustic waves [4] and robotic devices [6], [7] have been made to automate and manipulate crystals.” Sound waves have also been used to manipulate crystals, and some (or all) of these citations should be in the manuscript: [1]Sound waves used to arrange crystals in an ordered array: o
[1] Guo, F., Zhou, W., Li, P., Mao, Z., Yennawar, N.H., French, J.B. and Huang, T.J., 2015. Precise manipulation and patterning of protein crystals for macromolecular crystallography using surface acoustic waves. small, 11(23), pp.2733-2737.
[2] Sound waves used to harvest protein crystals for data collection in synchrotrons: o Cuttitta, C.M., Ericson, D.L., Scalia, A., Roessler, C.G., Teplitsky, E., Joshi, K., Campos, O., Agarwal, R., Allaire, M., Orville, A.M. and Sweet, R.M., 2015. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening. Acta Crystallographica Section D: Biological Crystallography, 71(1), pp.94-103.
[3] Sound waves used to deliver protein crystals to a FEL: o Roessler, C.G., Agarwal, R., Allaire, M., Alonso-Mori, R., Andi, B., Bachega, J.F., Bommer, M., Brewster, A.S., Browne, M.C., Chatterjee, R. and Cho, E., 2016. Acoustic injectors for drop-on-demand serial femtosecond crystallography. Structure, 24(4), pp.631-640.
These references have been added to the manuscript.
In terms of acoustic manipulation, the key innovation demonstrated in this manuscript is the first demonstration of manipulation of moving protein crystals. All other methods used to date have manipulated protein crystals that were initially at rest (sound waves were then used to impart momentum to the crystals, which then put them into motion). Given the enormous importance of jet technology to FEL crystal injection, the inability to acoustically manipulate moving crystals has been a crippling limitation in acoustic implementation at XFEL sources. This manuscript describes a highly important and likely paradigm shifting innovation, but this innovation has not taken place in a vacuum. Readers will be interested in the history of acoustic crystal manipulation, which will help put into context the key innovation demonstrated here: the manipulation of moving crystals, such as crystals within a FEL jet.
We agree with this and the above comments. The introduction has been substantially adapted to put more emphasis on the FEL application of our technology. In the results and discussion the following line has also been added to the manuscript (line 153-155):
‘This is especially of relevance in current serial crystallography experiments were a thin jet is brought in the X-ray beam.’
5. Methods/Results:
Line 82: Grammar, use “are” instead of “is”: “A schematic representation and a picture of the 82 acoustofluidic chip is are shown …”
This has been adapted in the manuscript (line 80).
Line 94: Grammar, use “were” instead of “was”: “A broad spectrum of biological compounds was were introduced…”
This has been adapted in the manuscript (line 91).
Line 101/105/122: Please be consistent in usage of chemical symbols or chemical names. If you are going to say “sodium acetate”, please also say “sodium chloride”. Note that it is fine to use acronyms for very long buffer names (eg MES, line 129).
This has been adapted in the manuscript (lines 101, 121).
Line 130: Readers who work on FEL problems will be very interested in the fluid velocity, rather than the volumetric flow rate.
The fluid velocity has been added were relevant (line 117 and 129).
Line 153: Crystal fowling is a HUGE problem in FEL injectors also. For some proteins, it is typical for only a few minutes of data to be obtained before the injector must be removed and cleaned, which can take as much as one hour to accomplish (the CXI station now features an automatic cleaning cycle, but it rarely works). This would be a good place to mention this huge advantage of combining the method described in this manuscript with FEL protein crystal injection, in preparation for a more thorough treatment in the discussion.
We agree with this comment. The following text has been added to the manuscript (line 185-189):
Crystals being focused in the center of a channel maintains their liquid mobility by preventing fouling to the surfaces. Fouling is a common showstopper in the pharmaceutical industry [30] but also in XFEL injectors [31]. Typically, only a few minutes of data collection can be done before the injector must be removed and cleaned. The use of bulk acoustic waves could not only allow efficient data acquisition but also prevent this enormous clogging problem.
6. Conclusion:
Line 212: “Increasing the local density of crystals is of great relevance for the current crystallography methods.” The sample utilization rate of XFEL injectors is very poor. High flow rate crystal injection is “thin”, such that the XFEL beam profile covers a significant portion of the crystal stream … but high flow rate injection causes the overwhelming majority of the crystals to be missed because they traverse the beam path between XFEL pulses (typical sample utilization rates are much less than 1%, and sometimes closer to 1 part per million). To avoid this problem, the sample injection must be greatly slowed using viscous media, for example using LCP, PEO, or agarose. However, viscous media cause the crystal injection to become very “thick”, usually around 100 um or more. For a 100 um crystal stream, a 1 um beam will miss 99% of the sample, hence again leading to very low sample utilization rates. If BAW methods can be used to arrange crystal samples within an FEL flowing injection stream, then please mention the remarkable potential savings in terms of sample utilization. Sample utilization is currently one of the biggest problems with the FEL solution to the structural biology problem. If you have a solution to this conundrum, please discuss it here. If not, it would be good to mention why not (for example, maybe it is not possible to arrange protein crystals within viscous media.
The conclusion has been adapted according to the comment (line 236-242):
‘We have shown the possibility to use standing bulk acoustic waves to manipulate flowing crystals inside microfluidic channels and have evoked the possibility to use this technology within a XFEL flowing injection stream. Current serial crystallography methods suffer from an extremely low efficiency, where the hit rate of the X-ray beam is typically less than 1%. Increasing the local density of crystals in a thin jet can lead to a much more efficient data acquisition. Moreover, the use of bulk acoustic waves could also prevent the enormous clogging problem of XFEL injectors. This technology could bring fundamental changes to the design of XFEL injectors. ‘
Reviewer 4 Report
Comments:
This paper reports a method for manipulation of crystals and high-density droplets by a standing acoustic wave in a microfluidic channel. This paper proposes a novel method that can be applied to analysis of microcrystals, and is worth to publish in Crystals with the following modifications.
1. Since the authors consider that their technique can be applied to the crystal structure analysis by FEL, they should refer to recent technical progresses on crystal sampling such as Acta Crystallographica D72, 520 (2016).
2. Reference 14 is not appropriate to explain Equation 1.
3. The authors should quantitatively show the focus thickness of the particles to the direction perpendicular to the flow and its dependence on the RF voltage and the particle sizes.
4. Figure 1a Frequence -> Frequency
5. The term, “radiation velocity” is not defined in the manuscript.
6. The references include wrong page numbers. The authors should correct them.
Author Response
We are grateful for the valuable comments of the reviewer, which were considered in the revised version of our paper as described hereunder. For the sake of clarity, reviewers’ comments are in black while our answers are in blue.
This paper reports a method for manipulation of crystals and high-density droplets by a standing acoustic wave in a microfluidic channel. This paper proposes a novel method that can be applied to analysis of microcrystals and is worth to publish in Crystals with the following modifications.
1. Since the authors consider that their technique can be applied to the crystal structure analysis by FEL, they should refer to recent technical progresses on crystal sampling such as Acta Crystallographica D72, 520 (2016).
This reference has been added to the manuscript (lines 34 and 39)
2. Reference 14 is not appropriate to explain Equation 1.
This has been changed to the following reference (line 134):
R. Barnkob, P. Augustsson, T. Laurell, and H. Bruus, “Acoustic radiation- and streaming-induced microparticle velocities determined by microparticle image velocimetry in an ultrasound symmetry plane,” Phys. Rev. E - Stat. Nonlinear, Soft Matter Phys., vol. 86, no. 5, pp. 1–11, 2012.
3. The authors should quantitatively show the focus thickness of the particles to the direction perpendicular to the flow and its dependence on the RF voltage and the particle sizes.
In the context of XFEL where we aim for a very narrow beam, this is indeed a very relevant question. The focus thickness is independent of the applied voltage. Only the velocity at which crystals migrate depends on the applied voltage. Theoretically, the node or anti-node is a point with a infinitesimally small width, so the jet thickness will be as big as the particles used. The smaller the particles the thinner the focused beam will be. We have performed experiments (unpublished) with relatively large crystals and the focused beam has approximately the same width as the size of these crystals.
No changes have been performed in the manuscript.
4. Figure 1a Frequence -> Frequency
The figure has been adapted in the manuscript.
5. The term, “radiation velocity” is not defined in the manuscript.
The following sentence has been added to the manuscript (line 58-59):
The migration velocity towards the pressure (anti)node is called the radiation velocity.
6. The references include wrong page numbers. The authors should correct them.
The whole reference list has been reviewed and adapted in the manuscript.
Round 2
Reviewer 2 Report
The authors still fail to address the issue of statistics in their two experiments. They only quantify a very small number of crystals in their experiments. They also only address the average size of focused crystals. I would like to see quantification of crystal/size across the width of the channel.
Another experiment that may improve the manuscript a lot is to actually take polystyrene beads and do the experiment in the simulation. A good correlation with their simulation will strengthen the value of this platform significantly.
As is I feel that their results fail to support their conclusion that the technology might pave the way to a novel type of XFEL injector.
Author Response
The authors would like to thank the reviewer once more for his evaluation and remarks.
The authors still fail to address the issue of statistics in their two experiments. They only quantify a very small number of crystals in their experiments. They also only address the average size of focused crystals. I would like to see quantification of crystal/size across the width of the channel.
For size measurements along the channel width a new experimental setup is needed which requires extensive additional engineering. As mentioned in the paper we will perform this type of measurements and separation of crystals of different sizes in a future paper. With the in-situ study we have performed and presented here, we can follow individual particles and study the migration behavior, no large crystal quantities are needed to support our findings.
Another experiment that may improve the manuscript a lot is to actually take polystyrene beads and do the experiment in the simulation. A good correlation with their simulation will strengthen the value of this platform significantly.
We have a manuscript currently in "a minor revision status" for a different journal that deals with fluorescent polystyrene particles. Moreover, the present paper is part of a dedicated issue of the journal on the crystallization of biological molecules (ICCBM19) for which we have specifically studied biological molecules.
As is I feel that their results fail to support their conclusion that the technology might pave the way to a novel type of XFEL injector.
We have emphasized this aspect as a reaction to the request of two referees (first review report) that wanted us to highlight the utility of our technique to the XFEL community in our revised version.
