High-resolution line-scan Brillouin microscopy for live imaging of mechanical properties during embryo development

Brillouin microscopy can assess mechanical properties of biological samples in a three-dimensional (3D), all-optical and hence non-contact fashion, but its weak signals often lead to long imaging times and require an illumination dosage harmful for living organisms. Here, we present a high-resolution line-scanning Brillouin microscope for multiplexed and hence fast 3D imaging of dynamic biological processes with low phototoxicity. The improved background suppression and resolution, in combination with fluorescence light-sheet imaging, enables the visualization of the mechanical properties of cells and tissues over space and time in living organism models such as fruit flies, ascidians and mouse embryos.

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Reviewers' Comments:
Reviewer #1: Remarks to the Author: The authors present a 780-nm line-scanning Brillouin microscope (LSBM) and demonstrate imaging of dynamical processes in living samples with low-phototoxicity. Although the developed microscope is technically impressive, it appears that LSBM cannot stand as a robust method on its own as simultaneous fluorescence guidance is necessary (because the Brillouin contrast is rather low as observed in all the LSBM images). While the need for fluorescence guidance may be acceptable though it will probably hinder the widespread use of the method, the spatial details provided by the Brillouin contrast are weak or absent, particularly when considering the corresponding spatial details of the fluorescence images or the high-resolution confocal Brillouin images of living organisms (e.g., https://doi.org/10.1364/BOE.10.001420). Due to the lack of spatial details, the quantification of the LSBM data is summarized by relative spatial means of the Brillouin frequency shift over large regions, which is not suitable for the high-resolution analysis of dynamic processes over space and time in living organisms. I am unable to recommend publication without the authors addressing the comments/questions below.
2. The phonons should be sketched with different size in the O-LSBM and E-LSBM schemes (Figs. 1c and 1d). How does the different size of the phonons and the spectral broadening of these two scattering geometries affect the Brillouin imaging resolution? These are important issues to discuss. 3. Were there artifacts in the Brillouin measurements owing to the single line illumination utilised? 4. Why are there wings in the O-LSBM PSF? Do the solid lines represent theoretical or numerical fits? This should clearly be indicated. 5. What are the factors that define the size of the focused line? How do the size of the focused line and the experimental spectral resolution of the instrument affect the mechanical resolution of LSBM? It is important to discuss this matter carefully. 6. The precision of E-LSBM seems to be better than that of O-LSBM. Is this a fundamental difference between the two scattering geometries? Why was the precision of E-LSBM measured at 532 nm? A good comparison requires the use of the same wavelength. The relative precision measured for E-LSBM seems to be twice as high as that measured for O-LSBM. How does this difference affect the ability to identify cellular and subcellular components? It is crucial to discuss all these issues, particularly in light of the work reported in https://doi.org/10.1364/BOE.10.001567. 7. Why is only the coarse structural mechanical dynamics detected by LSBM in Figs. 2c and 2f? Is it possible to significantly improve the spatial and mechanical resolution? Is there a fundamental limit to the spatial and mechanical resolution of LSBM? 8. Would an analysis of the spatial variance of the Brillouin frequency shift across the regions of interest in Figs. 2c and 2f result in additional insights about the investigated dynamics? 9. The scale bar in Figs. 2c and 2f is not clear and should be corrected. 10. What is the scale for sub-cellular components in the Phallusia mammillata? Can the spatial details in Figs. 3b and 3e be significantly improved by increasing the effective pixel time (to increase the SNR) and the spatial sampling/resolution? 11. Is the data in Fig. 3j statistically significance? Can the three Brillouin shift frequency components be identified distinctly from the entire image without segmentation? Why? 12. Were the Brillouin signals detected when imaging the living organisms also shot noise limited, as measured in water? According to which criteria was the effective pixel time of 1 ms chosen in the biological samples? 13. The time interval for calculating the illumination energy in SI Fig. 4d should be mentioned. 14. Characterization of the precision and accuracy of the LSBM spectrometer as a function of the spatial location in water is important (e.g., the interference between the spatial and spectral dimensions on the camera). 15. Why are the nuclei weakly/not detected by LSBM in SI Fig. 8b? Will a larger effective pixel time help? Can a confocal Brillouin microscope detect them safely? A detailed comparison between the imaging performance of confocal Brillouin microscopy and LSBM is required. 16. What will be the results of the experiment of SI Fig. 9 using 780-nm confocal Brillouin imaging? Where will phototoxicity-optimized 780-nm confocal Brillouin microscopy appear in SI Fig. 4d? will it overlap the operation region of LSBM? 17. Can LSBM with an effective pixel time of 1 ms pixel be shown to achieve high-resolution images of living organisms comparable to those of confocal Brillouin microscopy (e.g., https://doi.org/10.1364/BOE.10.001420)? 18. What is the maximum frame rate of the LSBM instrument? 19. If the analysis of the LSBM data comes down to the mean Brillouin frequency shift over the image (or over a few large, segmented regions of the image), why would not time lapsed imaging of a line in the sample by LSBM or multiple points in the sample by a 780-nm confocal Brillouin microscope with fluorescence guidance be adequate to study the samples presented in this work? This would enable to measure faster dynamics limited by the camera exposure time rather than by the volume imaging time of LSBM. Nevertheless, it should explicitly be noted in the manuscript that the fastest dynamics LSBM can probe is limited by the camera exposure time of ~100 ms rather than the effective pixel time. 20. Are 3 embryos an adequate sample size for the statistical analysis presented in Figs. 2d and 3j?
Reviewer #2: Remarks to the Author: A Summary of Key Results: The Brillouin microscope reported here uses line scanning and epi or orthogonal detection to increase image speed to circa 1 ms per voxel whilst achieving up to 180x165x170μm field-of-view (FOV) with down to 1.5μm spatial and down to 2min temporal resolution, whilst achieving circa 80 dB spectral extinction and a spectral precision of <20 MHz. The microscope also incorporates a SPIM setup for co-registered fluorescence imaging.
The key results in this work are three examples to show that Brillouin microscopy's capability in relation to temporal dynamics (live drosophila embryo), high-mechanical property resolution (the ascidian Phallusia mammillata), and low photo-toxicity (mouse embryo). I would have found a table summarising the experiment, including the mode and variable FOV and spatial resolution to be very helpful in sorting out the myriad details. Could this be included?
In epi mode at 20 mW illumination power is viable (no observed photodamage and 24-hr post viability) on live drosophila embryo over a time lapse of 30 minutes or so, at around 10 times lower illumination energy per pixel. Co-registered SPIM is included.
In orthogonal mode, the microscope was used to image the ascidian Phallusia mammillata over up to 14 hours -what was the illumination power of the laser? Co-registered SPIM is included.
And (can the mode be stated please -paragraph commencing line 172) with acquisition times of ~11-17min at 75-90min intervals over 46 hours, sensitive mouse embryos were shown to be undamaged, whereas, damaged when imaged using "conventional" confocal imaging parameters and wavelength. B Originality and significance: Line scanning in an orthogonal detection geometry was proposed and published in 2016 -Sci Reports: Ref 20. Can explicit reference be made to the novelty in the current manuscript? I expect this may be a collection of incremental improvements that collectively add up to a technically challenging and high-performance implementation -relative to previous incarnations, but this should be clearer from the manuscript. If the significance is to claim new mechanobiological capability, then this too should be clearly stated. Right now, it seems this manuscript would be well suited for Reviews of Scientific Instruments -a complex and highly capable instrument developed and described with exemplar samples and targets. The claimed advances need to be more specifically made and substantiated.
In lines 189-191, in summary, it is claimed "Compared to alternative Brillouin scattering approaches and implementations7,9,13,14,18 this represents a >20-fold improvement in terms of imaging speed, at >10fold lower illumination energy per pixel without sacrifices in measurement precision7." This is very clear, but it is not clear which specification is demonstrated by which experiment, i.e., whether they can be achieved simultaneously, and the claim of high mechanical property resolution of example 2, appears to be contradicted by the claim of without sacrificing measurement precision, when an improvement might have been expected.
Overall, it is indeed encouraging to see Brillouin microscopy continue to progress as a non-contact mechanobiological imaging technique -I am supportive of publication, once minor issues are addressed and subject to a convincing explanation of novelty and significance. C Data & methodology: There is a huge amount of detail in methods and supplemtnary information that broadly speaking is well presented and convincing. As indicated, a table summarising the three examples would help as there is a lot of change between experiments.
Can the authors spell out any negative consequences of incorporating SPIM, for example, on phototoxicity?
For the orthogonal mode, it would seem that there is potential for droop in Brillouin frequency versus illumination depth, arising from refraction, sample heterogeneity, and the Brillouin frequency shift's dependence on angle to the incident beam. Was this observed? If so, what was its magnitude? If not, why not? D Appropriate use of statistics: No specific comments. E Conclusions: Overall, this manuscript provides a comprehensive description of the instrument and the experiments performed using it, but the wood is lost for some extent to the trees -the advances in capability are less clear. See above for novelty and significance. As regards the experimental findings, can the conclusion/summary be clear on what has been seen before and what is reported here for the first time.
F Suggested improvements: experiments, data for possible revision: As above, greater clarity around what the novelty and significance is would help frame the manuscript as a pivotal one whereas at present this clarity is lost in the detail.
G References: Appropriate credit is given to previous work.
H Clarity and context: I found the abstract rather weak and did not convey the key novelty and achievements well at all.
Reviewer #3: Remarks to the Author: The manuscript "High-resolution line-scan Brillouin microscopy for live-imaging of mechanical properties during embryo development" by Bevilacqua et al propose a new Brillouin microscopy design, which brings a much-needed improvement to the technique. Indeed, combining line-scanning approach with near infra-red illumination, their proposed design allows for a faster less-toxic acquisition of Brillouin microscopy images without sacrificing measurement precision, a quality that is important for long term imaging live samples and especially dynamic processes. Furthermore, the microscope has a dual mode: an O-LSBM to allow for better axial resolution and lower phototoxicity, and an E-LSBM that minimises effects from scattering and optical aberrations (at the cost of lower axial resolution) and thus is suited for heterogenous samples. Finally, adding a GPU-accelerated routine for data visualization makes the microscopy technique more practical and insightful to the user, as it allows for in situ visualization of the data. The authors test their microscope in three different model organisms, exploring the potential applications of their system.
The manuscript is written clearly, and the appropriate controls and measures are conducted in most places and described in detail. There are however a few comments and suggestions that I believe would improve the quality of the manuscript. Overall, I believe that this manuscript will open the way for increased use of Brillouin microscopy in biological/biomedical studies, and would therefore recommend publication in Nature Methods following consideration of the points below.
Main comments/suggestions: Line 64-66: "Our microscope is based on a line-scanning approach that enables multiplexed signal acquisition, allowing the simultaneous sensing of hundreds of points and their spectra in parallel." Considering that 90° scattering geometry of LSBM broadens spectra and therefore limits resolution1, could the authors please comment on if/how they have overcome this limit? E-LSBM vs O-LSBM: From Fig 1f, it seems that the Brillouin Shifts measured by the E-LSBM are systematically higher than those measured by O-LSBM. Is this something expected from the system (e.g. from the optical geometry/setup). Furthermore, Fig SI4d shows that for the same illumination energy, the precision of O-LSBM is more than E-LSBM, hence being less toxic for live imaging. However this contradicts with Fig 1f, where it seems that although O-LSBM gives better spatial resolution, the precision of the E-LSBM is higher (smaller standard deviation). Could the authors please clarify this, for example perhaps different laser power/illumination energy was used when comparing the two modes in Fig 1f? Line 144-146: "Similar to the observations during VFF, the average Brillouin shift within cells engaged in tissue folding also increased during PM". The cells in the contractile region are probably those that move and deform most. I was wondering if the movement of cells could affect the light scattering and therefore the Brillouin shift? If so, then the increased Brillouin shift in this region might not necessarily be the result of the higher contractility but the result of higher movement? Could the authors please comment on this.
Line 147-149: "No photodamage or -toxicity was observed at <~20mW of average laser power, and viability assays showed that all embryos (n=3) imaged progressed to the first larval stage (24hpf)." The fact that all images embryos progressed to the first larval stage is indeed a strong indication of minimal photodamage. However, the authors could make their case even stronger if they showed that the imaged embryos developed into normal adults.
Line 157-160: "We observed a perinuclearly localized, high Brillouin signal within the B5.2 cells in the late 16-cell stage (Fig. 3e,f). This subcellular region is known to have a dense microtubule bundle structure driven by the centrosome attracting body (CAB) (Fig. 3d)." The authors use this example to show the ability of the O-LSBM to mechanically probe subcellular structures with high resolution, and this would suffice for the current manuscript. However, their result and manuscript would be much stronger if they could perturb the microtubule structure (for example with Nocodazole treatment) and show that the stiff region is gone, confirming that the high Brillouin shift region is indeed due to the dense microtubule bundle structure in these cells. Line 167-169: "These experiments also demonstrate that LSBM imaging can be used over long periods (here 14 hours; SI Video 3-4)". The authors indeed show that the organism is viable over long period of imaging. However, a more quantitative analysis (e.g. LIVE/DEAD assay or similar methods) would be desirable, especially because low phototoxicity is one of the main features of the proposed microscopy method.
Line 172-174: "Finally, to test the low-photo-toxicity of the LSBM approach in a further organism, we imaged the developing mouse embryo, from the 8-cell stage (E2.75) to the late-blastocyst (E4.5), covering a 46-hour time-span (SI Fig. 8)." The authors only show the initial and final timepoints of this experiment. It would be beneficial to include a movie of this process so the reader could see the intermediate timepoints as well.
Line 177-180: "Despite the embryos' notorious photo-sensitivity, no photodamage or -toxicity was observed at <~20mW of average laser power as confirmed by the morphology, dynamics, cell number and cell fate of the imaged embryos resembling those of control embryos (SI Fig. 8c,d)." Currently the authors show exemplary images of the imaged embryos and show that they are normal in terms of morphology and cell fate. It would be beneficial to include more quantitative measures of cell number and dynamics to confirm that the long-term imaging was non-toxic.
Minor comments: Line 324: Typo. "raise" should be "rise".  Thank you for submitting your revised manuscript "High-resolution line-scan Brillouin microscopy for live-imaging of mechanical properties during embryo development" (NMETH-BC48320A) and for your patience during the review process. It has now been seen by the original referees and their comments are below. The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Methods, pending minor revisions to satisfy the referees' final requests and to comply with our editorial and formatting guidelines.
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Reviewer #1 (Remarks to the Author):
In the revision, the authors addressed adequately the issue of sample photodamage, but addressed only to some extent my concerns regarding the spatial/spectral/mechanical resolution, precision, acquisition time, and fluorescence guidance in LSBM. It appears that the present LSBM system with 0.8-NA illumination and detection objectives and ~1-ms effective pixel time is appropriate for longitudinal imaging experiments in large samples but with limited 3D resolution and spatial averaging of the Brillouin shift, as discussed mainly in the supplementary material. I think that the main points of this discussion need to appear in the manuscript.
It is still unclear to me whether the cost for achieving sufficiently fast imaging speed in the selected samples was at the expense of a good mechanical contrast (and resolution), which looks weak (and blurred) in all the images and too pixelated in the zoomed images. No experimental evidence was provided on the effectiveness of LSBM for fast mechanical imaging with sub-micrometer resolution, which is important in many biological studies (e.g., cells). Thus, I think that claims about high resolution are misleading. Also, the statement about the benefit of resolving closely spaced Brillouin peaks in the probed volume is not fully accurate (see 10.1038/lsa.2017.139). The Editor will evaluate the fit of the manuscript to this high profile, broad interest journal.
Minor comments: 1. In the supplementary material it is stated that "We designed the thickness of the illumination line to be ~1μm, that is the typical mechanical resolution achievable in a biological sample.", where in the rebuttal letter it is written "We designed the size of the illumination line to be below ~2μm, that is the typical mechanical resolution in biological samples." Which statement is more correct? 2. The expression La=V*ΓB<sup>-1</sup> in the supplementary material should be corrected to La=V*(ΓB/2π)<sup>-1</sup> 3. The results in Figure 3h are confusing and a biological interpretation of these results would be helpful. 4. Cannot the E-LSBM path be slightly modified to provide also CBM at 780 nm? 5.3D render images of the data would be a valuable addition.
Reviewer #2 (Remarks to the Author): I observe that the authors have rigorously and comprehensively addressed the comments of three reviewers -my own to my complete satisfaction. In particular, the novelty has been clarified to my satisfaction. Extensive materials have been added to the Supplement which help clarify many details. My only advice would be to consider the number of significant figures in places -for example, in Figs 1e and 1f, are four significant figures really justified? I would ask the authors to review and consider this, here, and elsewhere.
Reviewer #3 (Remarks to the Author): The revised manuscript has been substantially improved with new supporting data/analyses that further clarify the method and biological conclusions. Sufficient major reviewers' concerns have been addressed, and we therefore recommend the paper for publication.

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