Enhanced Performance of the Optimized Dye CF583R in Direct Stochastic Optical Reconstruction Microscopy of Active Zones in Drosophila Melanogaster

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The use of super-resolution single-molecule localization microscopy (SMLM), particularly dSTORM, is limited by the problem of finding good combinations for multicolor imaging. This study compares the performance of a newly introduced rhodamine dye, CF583R, to established dyes AF647 and CF568 in imaging the presynaptic active zone scaffold protein Bruchpilot at Drosophila melanogaster neuromuscular junctions. The authors find that CF583R performs comparably to AF647 and is superior to CF568 in certain analyses, suggesting that the AF647/CF583R combination could be effective for future two-color dSTORM imaging of synaptic structures.

The authors are addressing a critical problem, namely that of multi-color imaging when using SMLM techniques. This issue is not limited to the specific organism or organelle they are studying, but of a larger concern for researchers in the field of cell biology.

The methods are described in detail, experiments well thought off and data analysed with care. I recommend the otherwise rarely detailed explanation for the statical method used.

The text is well written, conclusions supported by the data and references adequate.

I have no concerns and recommend for publication.

Author Response

We are very thankful to the reviewer for her/his careful evaluation of our work and its contribution to the field, as well as for recommending publication of our article.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript reported the study of the CF583R dye in dSTORM microscopy of the active zones at the Drosophila melanogaster neuromuscular junction. The results show that CF583R shows comparable performance as AF647 in AZ substructure analysis and it has better performance than the CF568 dye, exhibiting a promising potential for the future dSTORM applications. The following revision could be considered:

1. The author could evaluate the effect from the concentration of the dye CF583R as the concentration may affect the quality of the imaging.

2. The author could increase the number of the samples as a limited number may not give a general statistical outcome. The author could add more explanation on this problem.

3. The environmental factors like temperature, light intensity, etc. should be evaluated if this dye will be applied to future application.

Author Response

Please see the attachement.

Author Response File: Author Response.docx