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

Editorial  Note:  Parts  of  this  Peer  Review  File  have  been  redacted  as  indicated  to 
remove third-party material where no permission to publish could be obtained.

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

With recent advances in super-resolution microscopy, and the increasingly recognized importance 
of mitochondrial dynamics in mediating mitochondrial function, the manuscript by Yang et al., is 
timely. The work describes a novel fluorescent probe that should facilitate super resolution 
microscopy of mitochondria via STED. In particular, the ability to watch cristae dynamics during 
fission and fusion events is very exciting. While the STED images of mitochondrial cristae are 
certainly beautiful, and it appears that the new dye described here may be superior to those used 
in previous published work looking at mitochondrial cristae using STED, I do have some concerns 
with the manuscript that would need to be addressed. 

Specific Issues: 
The section describing how the Mito-ES1-635  dye labels mitochondria is confusing. The authors 
state that it ‘labels the plasma membrane with high density’. Then the authors say that the signal 
is ‘concentrated in the eccentric perinuclear regions’, but I’m not sure what they mean by these 
statements and how they are relevant to mitochondrial staining. Clearly the dye does co-localize to 
mitochondria based on the images shown, but the description in the text is not clear. 

The authors claim that the Mito-ES1-635  dye is covalently binding with mitochondrial VDPs. 
However, in Sup Fig 3, they show that MitoESq-635 binds to TRX-1, a cytosolic protein. Based on 
the evidence provided, it is unclear to me that the dye is in fact binding specifically mitochondrial 
membrane proteins, which I think is what the authors are trying to say. 

I’m not sure that I agree with the authors that there is no phototoxicity. They describe changes in 
the mitochondria, ‘bubble structures’, with cristae growing inside, which I would interpret as 
swelling of the mitochondria. In particular, the changes in fig 3 between the first frame and the 
final frame look like swelling and fragmentation of the mitochondria, which is you would expect to 
see with phototoxicity. If this was the ‘normal’ state of mitochondria, it should be evident from the 
start of the imaging, not just later on. 

This swelling also appears evident in Fig 4, as the width of the mitochondria appears to increase 
significantly over time, and not just due to fusion. While it appears that they can in fact catch 
fusion events, due to the swelling, I’m not sure how well the cristae dynamics they observe reflect 
what normally happens during fusion. 

In order to accurately compare the Mito-ES1-635  dye to other dyes used to image mitochondrial 
cristae, they should show their own data using their scope and settings, rather than pulling 
numbers from other publications, as settings and laser power are often unique to each microscope. 

General comments: 
I would completely remove the first paragraph, which I feel is unnecessary. The evolutionary 
history of mitochondria and their role in cancer are not really relevant to the current work. 

Though mutations in OPA1 cause an optic atrophy phenotype, OPA1 does not encode a 
photoreceptor, and this has nothing to do with sensitivity to light or phototoxicity. 

The authors need to include a bit more information regarding the deconvolution analysis they are 
applying to their images. 

Regarding the average widths of cristae they use 42nm and 43nm for the width following fission. I 
assume this should be the same number. 

The authors state ‘the fluorescent molecule could melt’, with high laser power. Not sure that ‘melt’ 
it the correct term here. 
 
While the images in Sup Fig 2 certainly look like mitochondrial networks, it would be nice to see 
co-localization with another mitochondrial marker for these other cell lines. 
 
Generally speaking, many of the images are too small to adequately see at 100% magnification. 
 
The co-localization images in Sup fig 1 are not great. 
 
For the supplementary videos 5 and 6, it would be nice to see the white dashed boxed areas 
shown in Fig 4 in order to focus the attention to the correct position. 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
The manuscript by Yang and coworkers addresses a timely need in super-resolution imaging, 
namely the generation of live-cell imaging compatible probes to follow mitochondrial dynamics. 
The topic per se is thus relevant and interesting to a wide readership. A very similar paper 
appeared in Proceedings of the National Academy of Sciences earlier this year (Wang et al., PNAS 
116, p15817 (2019)). In that paper, also a mitochondria-selective dye was presented for STED 
imaging, allowing observation of cristae dynamics. Therefore, claims of novelty such as “for the 
first time” in the abstract and elsewhere are problematic and that prior work should be 
acknowledged. Imaging in the far red as in the present manuscript (STED laser at 775 nm) is 
desirable to reduce interactions with cellular constituents. In terms of imaging performance, I 
would rate the PNAS paper higher (albeit at 660 nm STED wavelength) and I would also rate a 
recent paper by the Jakobs group higher in terms of imaging quality, using SNAP tag and silicon 
rhodamine for depletion at 775 nm (Stephan et al., Sci Rep 640920 (2019)). Therefore, the 
specific merit of the manuscript is to provide a label for STED microscopy at 775 nm which has 
affinity for mitochondria. 
However, the manuscript has several shortcomings, such that I cannot recommend it for 
publication in its present state. Several of the claims are not sufficiently supported by the quality 
of the data. In other instances the information provided is insufficient to truly assess the validity of 
the measurements. 
 
Please find my detailed assessment below. 
 
Major points: 
1) Absorption coefficient, quantum yield, fluorescence lifetime etc. for the squaraine dye should be 
given. 
2) Reporting of imaging parameters and timing of recordings is insufficient, making it either 
impossible to gauge the true merits or rendering statements misleading. E.g. the section on STED 
imaging in the Methods section states that each frame was obtained at 1 s and the image series 
were obtained at a 1-minute interval but it is not clear to which measurements this refers. Precise 
timing information is warranted in each image caption and detailed imaging parameters should be 
stated for each measurement. Similarly, all other experiments should be described in sufficient 
detail to reproduce them. 
3) In the abstract the authors state that live imaging over 50 minutess is possible. This is a 
meaningless statement if the actual time it takes for one frame and the intermittent (resting) 
times are not stated. As far as I understand, this statement refers to Suppl. Video 4, where each 
frame took about 4 seconds and then a recovery period of about 70 seconds was used, yielding 40 
images in total. The authors should rather state the number of images that were taken. 
4) The authors claim a resolution of 35.2 nm. This statement rests on a single line profile shown 
and a histogram without  any detail on how data was analysed. From the supplementary videos and 
the image material provided, I am very doubtful that signal-to-noise ratio is sufficient to claim 35 
nm resolution or even 35.2 nm. The authors are encouraged to refer to the relationship of 
resolution and signal-to-noise ratio in STED microscopy, discussed e.g. by the Vicidomini group 
(Tortarolo et al., Evaluating image resolution in stimulated emission depletion microscopy. Optica, 
5, p32 (2018)). Also the resolution determination in Fig. 2c is problematic due to insufficient 
signal-to-noise ratio. From most of the images provided, I find it hard to clearly delineate 
individual cristae. In comparison, cristae stand out very clearly in the Scientific Reports paper from 
the Jakobs group. 
5) I am somewhat concerned with the specificity of labeling for mitochondria. In Suppl. Fig. 1, 
there seems to be strong correlation with ER, which is, however, mentioned in the main text. 
6) p5: The description of Suppl. Fig. 6 in the main text implies that no toxicity arises during 1 h of 
STED imaging. However, in Suppl. Fig. 6 it is not clear whether STED imaging has been applied in 
addition to the incubation. It is not described what the viability assay is. 
7) p 5: The statement on saturation intensity is contradictory. This measurement is not described 
properly. From the data provided it is not clear that this measurement was done in a way that 
excludes e.g. the possibility that bleaching might influence the measurement outcome. While 
superior imaging performance over Atto647N would be an achievement, this statement would need 
to be supported by further data. 
8) p5: I find the wording on 3D imaging somewhat misleading. The authors apply only resolution 
enhancement in the focal plane, not along the optical axis. Taking a stack of 5 STED images with 
xy resolution enhancement and confocal resolution axially is not particularly challenging. 
Therefore, also the statement in the conclusion “STED imaging of 3D stacks can reveal the 
ultrastructure of mitochondria in live cells” is not justified for the measurements presented in the 
manuscript. 
9) p7: The authors observe formation of bubbles and fission of mitochondria in long-term STED 
imaging. It should be checked that these dynamics are not related to the photo-burden inflicted by 
the imaging. 
10) Suppl. Note 3: No data is shown to back up the claims that the squaraine dye has wide 
applicability. 
11) The Supplements, in particular Suppl. note 4 seem to be an unedited draft version with a 
series of problematic statements. For example, the statement on acquisition speed is not tenable 
in this unspecified way in view of the work by Schneider et al., Nature Methods 12, p827 (2015) 
where 125 frames per second super-resolution imaging rate was achieved. 
12) Suppl. Fig. 5: If MitoESq-635 is covalently linked to inner membrane proteins, it is no surprise 
that it stays bound also with prolonged imaging. This does, however, not imply that the 
mitochondrial membrane potential is till intact. 
 
Minor points: 
1) p2: Wording is unfortunate: “ATP, which is the only energy form that cells can absorb”. ATP is 
certainly the most prevalent energy carrier, but it’s not absorbed by the cells but rather 
synthetized from ADP. Also there are other high-energy bonds in cells that can be used to drive 
biochemical processes. 
2) p 2: I am not sure that mitochondrial dynamics “regulate” cancer cell activity. 
3) p2: The statement on the sampling theorem is unfortunate. Resolution is defined as the 
capability to discern two nearby structures. 
4) p2: There is an inconsistency with the author name in the citation Dannie et al.. 
5) Several of the citations are incomplete. 
6) p3: It would be helpful to include a statement on the absorption wavelength range of OPA1. 
7) p3: “Stimulated emission” is not a laser process. “Laser” means “light amplification by 
stimulated emission of radiation”. Stimulated emission is an important component of the laser 
process, but not all stimulated emission is equivalent to lasing. 
8) p3: “…the efficiency is generally too low to generate effective STED processing.” This sentence 
needs rewording. 
9) p4: I am not sure whether the authors want to state that they label the “plasma membrane”. 
10) Fig 1c: The authors plot power, not “intensity”, as stated in the figure caption. 
11) In Suppl. Fig. 1, the last column is not adequately described. 
12) p 8: What do the authors precisely mean by “melting” of dye molecules? 
13) Fig. 4: What is a “skeletal image”? 
14) The data in Suppl. Fig. 4 is consistent with covalent labeling, but not a “proof”. 
15) Suppl. note 1, line 66: “literature procedure” would warrant a citation. 
 
 


Reviewer #3 (Remarks to the Author): 
 
The paper titled “Mitochondrial dynamics quantitatively revealed by STED nanoscopy with an 
enhanced squaraine variant probe” reported a new fluorescence living cell probe，MitoESq 635. It 
is an excellent candidate for live cell STED nanoscopy, which will make real impacts on pushing 
super resolution microscopy towards practical live cell STED imaging. The manuscript is well 
written and timely. With much better performance over the existing mitochondrial probes, the 
authors obtained beautiful super resolution images of mitochondrial ultrastructure in 3D and long 
term (about 1 hour) using STED nanoscopy. They got mitochondrial cristae dynamical videos with 
a decent resolution of sub-50 nm for 1 hour for the first time in super resolution microscopy and 
mitochondrial research field. I have several minor questions for the authors to address before it is 
published on Nature Communications. 
1. In figure 1b, I suggest the authors to add the labels of excitation and STED wavelengths. 
2. In figure 2c, figure 3c and figure 4d, the details of how the data points were fitted should be 
described. 
3. In Supplementary Figure 9, all the scale bars on fluorescence images were missing. 
4. Although this probe worked pretty well with STED nanoscopy, the performance of this probe in 
live cell STED could be further improved if it can be combined with recent new STED technologies 
such as RESCUE STED, MINFIELD, DyMIN, STEDD, et al. Can the authors discuss about this？