FRET-Based Ca2+ Biosensor Single Cell Imaging Interrogated by High-Frequency Ultrasound
Round 1
Reviewer 1 Report
1. Figure 3 to Figure 6 need to add the ordinate label and unit.
2. In the "Discussion", the authors mostly describe what has been done and some of the current situation, which is not convincing enough. It is recommended to conduct a quantitative analysis based on the experimental results.
Author Response
Dear Reviewer,
Authors really appreciate your comments. We made every effort to address your critiques. Please see the attachment.
Thank you.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Yoon et al report on the development of a fluorescence resonance energy transfer (FRET)-based biosensor using EGFP and FusionRed fluorophores, for live cell imaging of Ca+2. The authors used 150 MHz ultrasound to mechanically stimulate individual cells, and showed that ultrasound is a credible localized alternative to chemical stimulation through ionomycin. They were also able to image transient Ca+2 spikes over time.
The manuscript is generally well-written and with clear aims. I was not entirely convinced by the claim that ultrasound exposure leads to both enhanced intracellular biosensor delivery and mechanical stimulation of Ca+2 channels. The authors did not provide data to discriminate the two effects, therefore I believe that this claim should be attenuated. If a direct membrane disruption is caused by the ultrasound wave, this should be shown, for example using a fluorophore which is typically membrane impermeable. That being said, an exhaustive list of limitations should be included in the discussion section, especially regarding the sensitivity and selectivity of the new FRET biosensor compared to existing compounds.
Nevertheless, I believe this manuscript is well suited for the journal Sensors and would be a useful addition to the FRET-bases biosensors field. I therefore recommend it for publication following extensive revision.
My detailed comments are given below:
Lines 16-17: How high? Please specify the precise center frequency used here.
Line 18: So HFU is used as a mechanical stimulator for specific subcellular regions? The transverse focus may be 10μm but the axial focal length should be much higher.
Line 20: Do the authors imply that sonoporation occurs due to HFU exposure? How is intracellular delivery enhanced through HFU exposure?
Lines 24-25: Please include the main quantitative results in the abstract.
Line 30: Please replace "molecules" with "molecule".
Lines 36-38: Please briefly discuss the FRET mechanism and why it's important in live cell imaging.
Lines 48-49: Do the authors mean the ratio of FRET acceptor intensity to donor intensity?
Line 49: At the donor fluorophore’s wavelength.
Line 61: Which other molecular event? Do authors refer to the intracellular Ca2+ steady state concentration, or Ca2+ exchange with the environment through ion channels?
Line 69: Please report the precise center frequency used here.
Line 70: Electronics "are" much cheaper...
Line 71: How is focusing on a specific cell achieved?
Line 80-84: This is confusing. Is cell membrane disruption intended or not in this setting? There must be acoustic parameters that induce a mechanical stimulus without disrupting the cell membrane. Do the fluorophores cross the cell membrane without ultrasound exposure?
Line 85: Please replace "and" with comma.
Line 95: You don't need to include the entire sequences in the main text. This can be shown as part of figure 1 or in the appendix.
Line 98: Start and stop codons are not shown in bold or italic.
Line 104: What is the molecular weight of the biosensor? Is it expected to cross the cell membrane?
Line 105: Ionomycin stimulation and fig. 1b are not mentioned in the main text. Also, excitation/emission wavelengths should be mentioned, with and without FRET.
Line 119: Is 1mm the focal length or the focal depth? Based on the schematic, it should be the focal depth. How long is the focus along the axial dimension?
Line 123: Excite the high frequency ultrasound transducer.
Lines 128-129: Is that to scale? The aperture size is 1mm and the focus ~10μm based on the authors remarks (although focal dimensions are not provided).
Line 133: Please fix to "transducer".
Line 143: Was the translation of the transducer coupled to the microscope lens? If not, how did authors assure alignment of the acoustic and optical focus?
Lines 148-149: Voltage values do not mean anything and are not translatable to other setups. Have the authors performed a calibration to measure the acoustic pressure at the focus?
Figure 3c: Please add a colormap label.
Figure 3d: How was the FRET normalized ratio calculated? Was it the average ratio within the entire cell or a specific ROI within the cell?
Line 181: SEM over how many measurements? How many cells were examined and/or how many excitations/cell?
Line 198: What sort of statistical analyses?
Line 220: So FRET-GFPRed does not require HFU exposure to transfect cells?
Line 245: HFU has been defined earlier, no need to repeat it here.
Lines 250-251: Authors should discuss Figure 5 in detail, e.g. the recovery dynamics of the FRET signal, the correlation with Ca+2 ion channel kinetics, etc.
Figure 4b-4d: The FRET/EGFP ratio increase after HFU exposure is around 16-17%, while the FRET/ECFP ratio is higher than 3fold. Why is there such a dramatic difference in sensitivity?
Line 283-284: It is not clear to me how the authors can discriminate between targeted delivery of the fluorophore and targeted stimulation using HFU. What is the difference between figure 4 and figure 6? Is there a negative control when HFU did not allow for targeted transfection but triggered Ca2+ flux or the inverse?
Line 330: HFU was defined earlier.
Line 331: This has not been convincingly shown.
Line 340: Calcium waves propagating across cells following sonoporation have been reported recently (Beekers, UMB, 2020). Authors should discuss the mechanotransductive effects of ultrasound exposure itself, and also, in relation to pore formation when using microbubbles to localize stress actuation.
Line 343: The question here, and even worse at 500MHz, is how to target a specific area of the cell. A feedback mechanism needs to be introduced, to make sure that the focus is at the right location.
Line 346: Please add an exhaustive list of limitations for the new FRET biosensor reported here.
Lines 359-361: This has not been deleted from the template.
Lines 365-370: Please add the contributions for this paper and delete the template guidelines.
Author Response
Dear Reviewer,
Authors really appreciate your comments.
We made every effort to address your critiques.
Please see the attachment.
Thank you.
Author Response File: Author Response.pdf
Reviewer 3 Report
The contents or order of Figures seems not to be properly arranged and hard to be understood (as well as the title of this manuscript). It is suggested that the author can add a brief title of each Figures to demonstrate the main theme of them. For example:
Figure 1: A is the design for new biosensor (name as: FRET-GFPRed, should be used throughout the whole manuscript including the Abstract for being easily read). B represents the sensing data of old sensor (It is not proper to change the original name of the well-known YC3.6 into any other name such as FRET-CFPYpet in this manuscript without any reasonable explanation. This makes the reader very confused!). In addition, the author should to put the representative plot of new sensor (FRET-GFPRed named by the author) in B, instead of that of YC3.6 (FRET-CFPYpet named by the author) to match the design shown in A.
Figure 2: In C (as well the descriptions from Methods), there is no further information to tell the reader how the author make simultaneously recording the two sensors in optical design (as the data shown in Figure 3E, F and others). While B and D displayed the spectrum and procedure of HFU for gene transfer and stimulation. However how to transfer gene and stimulate the cells is not described in detail in the Method. How long is the pulse, how many gene in the buffer, and when the cells will be ready for observation and for further experiments? Are the condition of HFU for both gene transfer and stimulation the same? If the condition is the same, can the author prove that this condition will not harmful to the cells?
Figure 3: The data present here are easily to be understood and make the reader confused with what sample were the author using, the new one (FRET-GFPRed) or the old one (YC3.6/FRET-CFPYpet)? The name of the sensor should be labeled on somewhere of the plot or cell images. Actually the data in C should be put in Figure 1B, since it is exactly the representative data of the new design Shown in Figure 1A. In E and F, again the same problem that the reader will be confused that what sensor did the two cells expressed (also Figure 4, 5, 6). In addition, the author should display the original fluorescent data (in G, R, C, and Y) and also label which cell is supposed to express (the new sensor or the old one) on the images.
Figure 5: The scale bar is missing. And again what sensor is used? Why use FRET/ECFP filter?
Figure 3 and 6 seems the same experiments, what is the differences between these two Figures?
Can't get the Supplementary Figures, so can't provide comments.
The main focus of this manuscript seems to be divided into two: one is the new sensor developed and the other is the novel applications of HFU (gene transfer and stimulation) combined with the new biosensor. However the weak point is that the first focus is not displayed very well, e.g. the benefit and the importance of FRET-GFPRed, compared to YC3.6/FRET-CFPYpet or other (in dynamic range or other aspect). And what is the purpose of using dual FRET imaging (including the benefits) for the following possible applications. The Methods also mentioned the traditional transfection using Lipofectamine 3000, how's different between HFU? What data came from the traditional one?
Ionomycin is not a physiological agent to generate calcium response, why used it, instead e.g. histamine or ATP?
On page 11 should be deleted: 11Authors should discuss the results and how they can be interpreted in perspective of previous studies and of the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.
Author Response
Dear Reviewer,
Authors really appreciate your comments.
We made every effort to address your critiques.
Please see the attachment.
Thank you.
Author Response File: Author Response.pdf
Reviewer 4 Report
This manuscript demonstrates FRET-based Ca2+ biosensor by single cell stimulation using high frequency ultrasound (HFU). Based on the previous achievement (Sci. Rep. 2016, 2017) in the same group, the current result showed that HFU-based transfection is achievable using two fluorescent protein couplers. In addition, it also exhibited HFU-driven calcium release in living cells. However, the following questions need to be addressed to be published in this journal.
- It should be explained whether HFU is capable of both gene delivery and protein delivery. And what are the advantages of using HFU over electroporation-based transfection in terms of delivery efficiency?
- In the material and method section, there was no information in protein expression and characterization of two different protein couplers (Figure 3A and 3B).
- In the material and method section, there was no information on the amount of used plasmid gene for HFU-based transfection (Figure 2).
- There was no discussion on the comparison of Figures 4 and 6, in terms of transfection efficiency between lipofectamine and HFU.
Author Response
Dear Reviewer,Authors really appreciate your comments.
We made every effort to address your critiques.
Please see the attachment.
Thank you.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The authors addressed all my comments. I am happy to recommend the manuscript to be accepted in its current form.
Reviewer 3 Report
No further comments.
