Peer review of: Mitochondrial dynamics quantitatively revealed by STED nanoscopy with an enhanced squaraine (2019)


RR2
Reviewers' comments:
Reviewer #1 (Remarks to the Author):
  Overall, the data and images in the manuscript show that the MitoESq-635 dye is a valuable tool
 for imaging mitochondria via STED microscopy. Yang et al. have made several key changes that
 help improve the manuscript. However, while the response the reviews was quite thorough, I did
 not feel the revised version completely addressed all of the key points discussed. Moreover, in my
 opinion some of the claims in the paper are still overstated. For example, there is clearly
 photobleaching which occurs over time, and there is clearly phototoxicity with the mitochondrial
 morphology and cristae structure changing over time. This will likely be true for any dye and does
 not detract from the paper, but to claim otherwise is misleading. What is valuable is the direct side
 by side comparison between the new dye and existing options, which clearly demonstrate the
 superiority of MitoEsq-635 (e.g. Sup fig 7). Perhaps some of this data could be moved to data in
 the main paper rather than supplemental.
 Specific Issues:
 Additional details for the imaging over 50 minutes are required in the abstract.
 Be careful about the specificity of various statements. The following is not true:
 “conventional optical microscopy techniques are insufficient to visualize the structure and
 dynamics of mitochondria”
 Generally speaking, researchers have been using conventional microscopy for nearly two decades
 to study mitochondrial structure and dynamics. However, if you are referring to sub-mitochondrial
 structures (cristae) being imaged live, the sentence would be more appropriate.
 Not sure how this sentence fits in the context of a paragraph on microscopy
 “The mitochondrial intermembrane space can be measured with indirect approaches such as
 proteomic mapping”
 The following statement is still false: “Mitochondria are more sensitive to light than other cellular
 organelles due to the existence of photoreceptors”
 Mitochondria do not have photoreceptors. Photoreceptors are a cell type, which have mitochondria.
 Furthermore, this is irrelevant to mitochondrial sensitivity to light.
 The text is still unclear with reference to the dye labeling the plasma membrane. The plasma
 membrane is the membrane at the exterior of the cell and is distinct from the mitochondria and
 mitochondrial membranes. Thus, it is unclear how the dye is binding to the plasma membrane and
 labelling mitochondria. Perhaps the authors mean the dye can bind membranes generally, rather
 than the plasma membrane specifically?
 The following sentence is poorly constructed: “Benefitted from its low saturation intensity, the
 MitoESq635 probe exhibited no marked toxicity in HeLa cells at a concentration of 1 μM after 1
 hour of incubation during STED superresolution imaging microscopy”. However, the bigger issue is
 that details on the imaging conditions are still absent, making the statement useless.
 While the following statement does have some key information, it is still incomplete: “With
 MitoESq-635, rare photobleaching and mitochondrial shape variations are observed upon exposure
 to STED scanning for over 10 minutes (1.2 s per frame, 700 × 700 pixels, 12.6 μm × 12.6 μm,
 STED beam of 30.2 mW at 775 nm), as shown in Supplementary Figure 7”. The reader needs to
 know how many images were captured over the 10 minutes. Is this one image every minute (e.g.
 a 1 minute recovery), or one image every 1.2 seconds (e.g. constant imaging).
 In the context of the Z-axis, how relevant are steps of 200nm, especially given that cristae are 30-
 50 nm in width?
 
Please clarify what the red and green colors in Fig 4i represent.
 For sup fig 7, be consistent with the labeling of MitoESq-635. In one panel it is the blue data points
 on the graph, and in the next panel it is the orange data points. Can these graphs be combined?
 Again, details matter, there are different details given for the MitoEs1-635 vs Mitotracker Green,
 than for the MitoESq-635 vs Mitotracker FarRed (e.g. dye concentrations). Why are the
 concentrations different for MitoESq-635 and Mitotracker FarRed, is this still a valid comparison?
 There are still several small typos/formatting issues throughout that required correction. For
 example, “standard gold live cell” should be “gold standard live cell”
 Reviewer #2 (Remarks to the Author):
 The authors have made improvements to the manuscript. However, they have glossed over
 several of the reviewers’ comments and have given explanations to reviewers without fully
 addressing the actual issues in the text. The paper is still not exact enough in many aspects,
 especially concerning imaging. I am still reluctant to recommend publication.
 Please find some specific points below.
 1) The authors have not removed some of the statements that I find problematic from the abstract
 and elsewhere, such as 35.2 nm resolution and “for long periods of time with minimal
 phototoxicity.” In fact, phototoxicity seems to be a problem, as mitochondria do display marked
 swelling. The authors now quantify swelling of mitochondria. Statements such as “showed healthy
 behavior with few changed to spheroidicity” might still be perceived as downplaying potential
 phototoxicity, especially as in the discussion, the other super-resolution imaging options for
 mitochondria are called out as being too phototoxic. Also in the present paper there seems to be a
 substantial light effect on the mitochondria and it should be discussed more fully that much of the
 observed alterations/dynamics could simply be due to phototoxicity. It should also be noted that
 phototoxicity is not just dependent on laser power. A large portion of it is mediated by fluorophore
 bleaching, such that the bleaching rate and dye concentration are also important parameters for
 phototoxic responses.
 2) The paper by Wang et al. in PNAS should be acknowledged in the main text and the specific
 merits of the two approaches discussed. It is not sufficient to cite it as a supplementary reference.
 3) I pointed out in my previous comments that signal-to-noise ratio is an important factor when
 determining resolution. E.g. the resolution measurement in Frame 60 of Fig. 3 is not meaningful.
 Signa-to-noise ratio in Fig. 2c is too low to perform a reliable resolution measurement on it.
 4) Fig. 1c: On what sample is the saturation intensity measured? How is such a measurement
 taken precisely? The authors state in the rebuttal letter that they cannot exclude that bleaching
 contributes to the apparently reduced saturation intensity of their probe vs. Atto 647N. Without a
 proper description of the procedures, it is not clear what the merit of such a measurement is and
 whether the conclusions drawn are valid.
 5) Reviewer #1 asked for a description of deconvolution. It is not helpful if this is in the response
 to the reviewer and does not enter the methods section.
 6) “Plasma membrane” is the membrane that separates the cell interior from the outside. From the
 authors’ explanation in the rebuttal letter I am still not sure they want to state that they label the
 plasma membrane rather than a mitochondrial membrane.
 7) Power and intensity in Fig.1 are still mixed up. Milliwatts are not a unit for intensity. This also
 applies to the saturation intensities stated.
 8) Can the authors please state how they assess viability (Suppl. Fig. 8, see my comment in the
 original referee report)? The data in Suppl. Fig. 8 is meaningless if the authors don’t give the
 
details on how they perform such an analysis.
 9) The new section on data processing is not understandable.
 10) The caption in suppl. fig. 9 and 10 does not explain how cells were labelled. It would be good
 to have such information directly in each figure caption.
 11) Suppl. Fig. 14: I cannot read the axes in panels d and e.
 12) Whenever a nonlinear lookup table is given, a colour bar for the lookup table should be
 provided. This also applies to the 2D histograms in suppl. figs. 1-3.
 13) Suppl. Fig. 2: It appears that slightly different focus positions are imaged in the two colour
 channels, lowering overlap.
 14) Hemozoin is a crystalline substance, such that “melting” is more appropriate there than for
 dye molecules.
 Reviewer #3 (Remarks to the Author):
After revision, I recommend this manuscript to be published.