Peer review of: Fast and multiplexed superresolution imaging with DNA-PAINT-ERS (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
Civitci et al. report an effort to improve the imaging speed of DNA-PAINT by the addition of 
ethylene carbonate (EC) into the imaging buffer. EC helped increase the unbinding speed of the 
imaging strand from the docking strand, and with the addition of repeating sequences and PEG 
spacers in the docking strand, they increased the binding kinetics. Thus they achieved DNA-PAINT 
with improved speeds at several minutes per image, and demonstrated two-color imaging. 
 
The work is generally interesting, and I recommend the publication of this work in Nature 
Communications, with the below comments. 
 
1. The authors should describe what their method is in the abstract. Now the name “DNA-PAINT-
ERS” just comes up from nowhere. 
 
2. What are the actual frame rates used to record the different data? Some of this information is 
hidden in the captions of their videos, but this should be more clearly stated in the text, captions, 
and the method section. For example, as they show good DNA-PAINT images in 150 s in Fig. 2a, 
how many frames were accumulated? If the frame rate was 67 frames/s as stated in the video 
caption, then 10,000 frame seems a bit low for typical SMLM? Also, in the videos shown, please 
add in timestamps and scale bars in each frame. 
 
3. In figure 1B, bottom panel, for the differential images, can the authors show both positive and 
negative changes in signal, e.g., using different colors? Then it could show which single molecules 
have unbound. 
 
4. The comparison of with and without  EC in Fig. 3AB may not be very fair, since in this case it was 
with a docking strain optimized for performance in the presence of EC. I suggest either moving 
them to supplement or be more cautious in the discussion. 
 
5. It would be useful to add in data to show whether the single-molecule brightness (photon count 
per unit time) is varied due to the addition of EC into the buffer. Fig. S1 alludes to this, but it 
would be useful to get histograms of photon counts. 
 
6. In the introduction part, “other SMLM (t on ~0.1 s)6”: 10 ms time scale may be more common 
for STORM/PALM. 
 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
in this work, the authors propose and test a number of technical improvements for DNA-PAINT, an 
approach to single molecule localisation microscopy that is based on the controlled, transient 
hybridisation of dye-coupled DNA oligomers with their reverse-complementary counterparts that 
are bound to a traget structure. This technique is promising as it allows for multiplexed labelling 
and higher resolution than other approaches such as (d) STORM and PALM. 
The authors present: 
 
a) an optimisation of the binding period for shorter, but equally specific binding events using 
addition of ethylene-carbonate, a reagent known from other DNA-based techniques such as FISH. 
The authors present very convincing, high quality data to support their claim that this speeds up 
DNA-PAINT imaging. 
 

b) a multiplication of the target sequence on the DNA to increase local concentration and faster 
on-rate. This significantly increases signal, although the quantification is not entirely clear and 
more controlled quantitative data, maybe acquired in vitro should be presented. 
 
c) the introduction of a polyethylene glycol linker between the antibody and the oligomer to make 
the oligomer more accessible. This furthermore increases the signal. 
 
Overall the authors present three tweaks to DNA-PAINT that will make this technique more 
efficient and accessible to many and this is an important and worthwhile contribution. The 
experimental setup is designed nicely and the results convincing. The only problem is that the 
quantification for b) and c) is not clear and should be demonstrated using a clear in vitro setup 
rather than intrinsically uneven biological images. 
 
I thus suggest minor revisions.