Peer review of: Spatially selective manipulation of cells with single-beam acoustical tweezers (2020)

Reviewers' Comments: 
Reviewer #1: 
Remarks to the Author: 

This paper presents an important step beyond that in [8] in which the manipulation of inanimate 
150um particles was shown with a 4MHz vortex device. The same device design principles are 
used here, but in the present paper a 45MHz device is shown. The extension is non-trivial, 
requiring enhancements of most detailed aspects of the design. Beyond the device manufacture 
difficulties, the present device is important as it gives access to selective cell manipulation. So, 
whilst the device in [8] was scientifically interesting, the present device is practically interesting. 
Indeed, although the present device was very much predictable from [8], it is nonetheless 
exhilarating to see it working so well – the videos make this particularly clear. The forces applied 
to cells and acoustic power are nicely analysed and quantified. Also, the heating effect is measured 
and found to be only a few degrees. These steps to characterise the device show it to be a 
potential competitor for optical tweezers, again, making this an important step. Initial cell viability 
has been assessed and looks promising, although cell proliferation post sonication was not shown. 
In summary, this is an important piece of work and should be rapidly published. 
 
Prior to publication, the following point should be considered. 
 
Line 38 – the power levels in medical imaging are much lower than here, so don’t think this is 
relevant. Hence, this discussion should be re-worded, or some precise quantification used. 
Line 43 – the attenuation of ultrasound varies strongly with frequency. It does not seem 
reasonable to say that its uniformly “weak” across this whole frequency range. Indeed, this 
statement raises the obvious question as to what is the upper operating frequency of this type of 
device? This would be interesting to explore in the discussion. 
Line 51 – the authors need to comment on the microbeam tweezers of Shung at USC, e.g. doi: 
10.1002/bit.23073. These operate in the ray-acoustic regime. I suggest the relative merits of the 
microbeam approach vs. the vortex are also mentioned briefly in the discussion. 
Line 188 – clarify that the field is sinusoidal in form and the quoted displacements are the 
amplitude of this wave. 
Line 193 – can the authors state the energy efficiency of the device, i.e. how much electrical 
power was input to generate this acoustic power? 
Line 258 – what is the closest approach of the manipulated cell with respect to another static cell – 
this defines the spatial selectivity. Can two touching cell be separated? 
Line 284 – why is this difference (between optical and acoustic tweezers) only one order of 
magnitude. If one looks at the difference in velocity of sound relative to light, one would expect a 
much larger improvement (5 orders of magnitude). Presumably the difference is due to the shape 
of the field used here relative to that used in optical tweezing, i.e. the acoustic field extends 
around and away from the cell, in the present device, whereas in optical trapping the energy is 
focused on the particle. 
Line 335 – would be better to say, “apparently harmless”, as a full viability study is not in place 
yet. But I agree that the results are extremely encouraging. 
Overall/discussion – can the authors comment on the range of cell-sizes and inanimate object 
sizes trapped with the present device. This would help readers understand the capabilities. If this 
is not known, it would also be worth stating this. 
 
 
 
Reviewer #2: 
Remarks to the Author: 

The authors report upon an ultrasonic methods for a “precise selective contactless manipulation 
and positioning of human cells in a standard microscopy environment, without altering their 
viability.” 
 
Major issues: 
 
1. The paper uses terms like – “cell selective manipulation”, “precise and selective” and “without 
altering their viability”, which to a biologist have specific meanings. I don’t believe the paper 
evidences these claims. The authors must provide detailed quantitative information showing 
precise, cell selective separation. Presently, the paper only shows simple 2D manipulation of small 
numbers of cells. 
 
The term “without altering viability” can have many meanings and at the least needs controlled 
data, although really data is needed showing that mechanosensitive receptors are not activitated 
(see later). 
 
2. The authors have already published all of the different technical abilities described in this 
publication in previous papers (e.g. tweezing and wavefront shaping). The novelty here is 
therefore low technically, the authors have simply increased the frequency with little engineering 
challenge. In truth they just show 2D patterning of cells which has been demonstrated acoustically 
and optically, or electro-optically. 
 
The merit in publishing this work will only arise through the context of the technique’s 
performance in enabling a clear analytical application for quantitative cell screening (point 1 
above), which currently could not be achieved using other acoustic, flow or optical techniques. 
Using the technique to place a few cells is incremental. 
 
Technical issues (all must be addressed in detail) 
 
1. Epoxy is a viscoelastic material and will heat up due to the acoustic wave propagation. I am not 
convinced that most of the heat is generated is due to the Joule heating. When expoxy is used to 
stick a glass slide to an ultrasonic transducer, the polymer will heat up considerably, in particular 
at the frequencies and powers used. 
 
2. On the subject of acoustic frequency, data needs to be shown quantitatively as why the 
frequency chosen was used. For example, why not 60 MHz or 80 MHz? 
 
3. The authors do not consider possible resonance frequencies of cells and related biophysical 
processes; for example, the response of suspended chondrocytes over a range of frequencies 
using continuous ultrasound has a primary resonant frequency of 5.2 ± 0.8 MHz 
(https://doi.org/10.1371/journal.pone.0181717). The author’s statement is not corroborated or 
evidenced. 
 
Cells are highly variable depending upon their history and their current environment. There is no 
evidence that the cells used were in anyway “controlled” in their state (biological, cycle, etc). 
Mechanical stimulation through the acoustic forces, even in the lifetime of the experiment will alter 
the cell biochemistry and biophysics. This needs discussion. 
 
Specific Comments (all must be answered) 
 
1. Abstract: "2 mW of driving power" - is this the acoustic power? 
2. Page 1, Column 2, Lines 41-45: 
The Frequency range is very subjective - it can go up to 1 GHz or more; 
3. Unsupported claim: "the weak attenuation of sound in both water and tissues at these 
frequencies limits absorption induced thermal heating"; This needs to be clearly referenced or 
demonstrated experimentally in ESI. 
4. Page 3, Column 1, Lines 134-135: delete "remote". It’s not remote. 
5. Page 3, Column 1, Lines 159-161: unsupported claim "poor thermal conductivity of glass". 
Relative to what, Water? Air? I think not. 
6. Page 7, Column 1, Line 399: "slow sound speed" - it is a subjective claim. Again, as above. 
Water?, Air? 
7. Page 7, Column 1, Line 383: "The first transducer was designed to excite transverse wave" - 
how does the transverse wave couples to fluid with cells? 
8. Page 7, Column 1, Line 397: Why mention a "transverse wave sound speed" transducer, if it is 
not used in this study? 
9. Page 8, Column 1, Lines 518-521: authors state: "For the determination of the amplitude of 
vibration, the tweezers were excited with the exactly same frequency generator (IFR 2023 A) and 
amplifier (AR Amplifier 50 W) as the one used in the experiments." - what was the output power 
from the generator to the amplifier? 
10. Fig. 6: Amplitude must be replaced by magnitude in both parts of the illustration. 
 
 
 
Reviewer #3: 
Remarks to the Author: 

This well-written manuscript describes a new kind of acoustical tweezer designed to capture single 
cells. The main experimental novelty is the miniaturisation of a 4.4 MHz acoustic tweezer to 47 
MHz frequency, which insures a much smaller precision. An acoustical vortex generates a trap 
whose size is such that a maximum force occurs 20 micrometers away from the trap center, and 
then the tweezer force vanishes. 
Experiments confirm the ability to move single cells among other ones that remain untouched. The 
manuscript is containing extensive confirmation of the efficiency of the trap, with measurements of 
the actual sound field, temperature increase measurements, drag force measurements and even 
cell viability tests. Numerical simulations show the trapping ability and the spatial profile of the 
sound field. 
Overall, this study is opening new realms for acoustic manipulation with increased precision, and 
brings physical evidence on the mechanisms at play. I suggest publishing the manuscript provided 
corrections are incorporated to address the following issues on the description of the tweezer: 
1. Line 31. « But, the dramatically lower speed of sound compared to light leads to driving power 
several orders of magnitude smaller. ». The term « dramatically» sounds strange here, with a 
negative side, that does not fit the positive side. 
2. Line 131. Figures are not called in order in the text, which creates confusion, since the reader 
has to search information in an apparent random order. Please fix this issue in other places of the 
manuscript, by changing the text or the figure order. 
3. Figure 2C: what is exactly the scale for the measured displacement? 
4. Figure 2E. Focalisation occurs at a z position that seems higher than the 1.1mm of the 
experiments. Please explain the implications of this difference. 
5. Figure 2F. Is the numerical prediction for the trapping force in agreement with the experiments? 
The manuscript is not really clear on this aspect. 
6. There is a non negligible pushing force in between 50 and 70 micrometers. Did the author 
observe such a repulsion force when dragging a cell nearby another that is very close? 
7. What would happen if the density of cells would be very important, with distances between cells 
less than 20 micrometers? 
8. Not much is said on the axial acoustic force. In principle it could push away the cell from the 
substrate, or attract the cell, depending on the actual position of the focus of the transducer. 
Please comment on this aspect.