Talk:Single-molecule FRET

We will be adding 3 major things to this article. Firstly, we will be expanding upon the existing methodology. Secondly, pictures will be added to various sections. Finally, an "Applications" section will be added which will include how smFRET can be used to gain further understanding of the structural dynamics of biomolecules, especially nucleic acids. Magnus Ma (talk) 01:41, 12 October 2016 (UTC)

Peer Review by Jingru (Group 1)

Overall it is a good extension of the previous wiki article, including limitations and more information on applications. Introduction is brief but concise and does the work, the reader could understand what smFRET does in the very first sentence. Methodology has a good beginning and make it clear the difference between two methods of smFRET. The advantage section is well organized and successfully addressed the difference between ensemble FRET and smFRET. And the applications and limitations are also included in the article, making it comprehensive and help the reader understand.

Methodology should be more organized

Methodology should be more organized in a sense that freely-diffusing and surface-immobilized methods are well separated and not mixed with the detailed techniques used by smFRET - it is sometimes hard to read if the paragraph is always jumping back and forth from one method to another.

In the applications section

In the applications section, one of the paragraph is actually the application of FRET. Also the author mentioned the benefit of smFRET to FRET, which should be included in the advantage section.

As for the application of smFRET, smFRET has been widely used in biological studies. As a Wikipedia article, there should be a a wider range of brief introduction of various applications. The author should make an effort to cover as wide as possible the applications in different fields, rather than using a majority of the section to describe one case of application (KirBac potassium channel for two paragraphs) in very details.

Other suggestions

More graphs, like a graph to illustrate the real experiment settings, the comparison of (simulated or real-experimented) signals taken from both methods of smFRET, etc., will largely help the reader understand the method. You can obtain some of these graphs for free on websites or from researchers.

Also, there is a comprehensive review on smFRET at http://article.sapub.org/10.5923.s.biophysics.201311.02.html which might help.

Cherrysu (talk)

67.194.230.56 (talk) 04:17, 21 October 2016 (UTC)

Peer Review from Group 1 (Shiba)

General comments: The article has been well modified by adding a lot of useful contents (advantages, applications and limitations) then the existing one. The contents represent a coherent flow of idea and illustrations. There are some specific things that the group might need to consider to make the article even more helpful to the general public.

Specific comments: 1) The group can make some significant change to revise the methodology section, which basically mentions about TIRF (wide field microscope) as a major tool for smFRET detection. There are other methods like confocal microscopy (which is briefly mentioned while mentioning about camera). There are no references to support the given statement about confocal microscopy for single molecule detection. There are few reports (attached below) those can be looked through and added to the methodology sections with proper citations. (a) doi: 10.1063/1.1589587 (b) Dennis R. Livesay (ed.), Protein Dynamics: Methods and Protocols (Chapter 3), Methods in Molecular Biology, vol. 1084, (DOI: 10.1007/978-1-62703-658-0_3)

2) There are also very useful single molecule techniques called fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), which has been used to look at smFRE T and can be discussed briefly in methodology.

3) One more advantage of smFRET is “other than looking at specific states, SmFRET is also used to look at intermediate conformations, which is otherwise hidden in ensemble measure. This gives a lot of important information about the evolution of different states with time trajectory. “ (Ref: Fluorescence Spectroscopy of Single Biomolecules, Shimon Weiss)

4) Ensemble FRET is mostly restricted to two-color systems, where one is donor and another is acceptor. However, smFRET have been successfully used for more than two color systems (three color, four color, etc). This is an advantage as well as a great application for looking at complex biophysical processes. (Ref: 10.1529/biophysj.104.043935, 10.1038/nmeth.1208)

5) One of the reasons single molecule FRET is popular over ensemble FRET, because they have been used successfully for looking at various biophysical/biochemical processes in live cell and study these behaviors over time.

6) SmFRET has a lot of applications in looking at the conformational change of surface bound as well as freely diffused molecules/biomolecules. So, I feel like the last sentence in the “Advantage section of smFRET” is kind of incomplete. The group may add some specific examples to justify this statement.

7) The “Application” section need a lot of key references (like at Ha et.al., Weiss et.al.). There are many illustrations for smFRET as a tool for looking at different biophysical processes, among which protein folding is one. For example, application in protein folding (one of which I mentioned above), looking at conformational changes in nucleic acid based systems (RNA folding, DNA folding), looking at lipid dynamics,etc. It has also been used to look at complex biophysical processes like protein-protein interactions, DNA-protein interactions, lipid-protein interactions, RNA-Protein interactions, in enzymology, etc. There are many articles available to corroborate these processes. The group may incorporate these references for the article.

8) Proper citations are needed for the conformational change study with ion channel motifs.

9) While the “Limitations” paragraph just focuses on one aspect which is distance, there are many other limitations to smFRET mentioned in various reports. Some of them are efficient labeling of systems with fluorophores (smFRET is insensitive to incomplete labeling), low efficient FRET pairs, photo-bleaching of a single fluorophore with time, oxygen-induced fluorescence quenching, limited time resolution by CCD camera (up to 1 ms making it less efficient to study faster dynamic processes). However, there are also various alternatives used by different researchers to tackle down these problems. The editor may corporate these specific aspects to make this article more helpful.

Overall, the wiki edit draft looks really good, except few minor flaws which can be addressed by the group. There are two great reviews by Ha et. al. (attached below) on smFRET which can be helpful for the group to add a little more contents to the article make it better. (10.1146/annurev.biochem.77.070606.101543, 10.1038/NMETH.1208) Ssd91 (talk) 04:20, 21 October 2016 (UTC)

Peer Review 10/20 G1 Erin R

Peer Review 10/20 G1 Erin R

The overall content of the introductory portion was satisfactory. As someone with no background knowledge of this subject, I could comprehend what was being said, but extra information about FRET technology in general and the difference between 'regular' FRET and smFRET may have been helpful. Also, I would recommend defining terms you will be using throughout the article early on. For example, ‘FRET efficiencies’ and ‘channel motifs’ could be expanded upon.

Each of the subheadings was of satisfactory length, although the unedited Methodology portion was a bit lengthy. It may be possible to have two subcategories of methodology, one for surface immobilized molecules and one for freely-diffusing molecules.

As you continue to edit this page, I would add more links to other Wikipedia articles, such as 'RNA folding,' 'native state,' 'dynamic temporal resolution,' and 'accumulation' in Advantages of FRET and 'KirBac potassium channels,' 'Force probe techniques,' and 'fluorophores' in Applications.

The example of channel motifs presented in the Applications was useful in visualizing the purpose of smFRET, but I recommend reorganizing to present the goal of the study first and then later highlighting how smFRET technology is useful in this scenario. The content of the example is good, I liked that there was a good explanation as to why smFRET was used over the averaged crystal structure method. I thought that the limitations section could use an example as well as this was a very broad overview.

The figure illustrated in this article helps visualize the importance of using a method, which is more specific than 'averaging,' however, the image could be polished to look a little more professional. Also, if it is possible, it could be beneficial to add example conformations of molecules to assign to each peak to understand what 'averaging' looks like in a molecular sense. Also, since this is an article about an imaging method, adding one of those images would help the reader comprehend what they are learning about (or a FRET population table). Another helpful figure could be an equation used to mathematically visualize 'distances between molecules as a function against time.' References are complete and the ones chosen show an array of source types, like journal articles and a book.

Overall, I think the article does a good job illustrating the importance of this technology in science today. The sections Advantages of smFRET and Applications contain some repeated ideas, like the imaging of averaged vs transition compositions of molecules. I would review these sections together and find a way to remove redundancy. The form could also be changed so that it reads less like a paper and more like a wiki article. Transition phrases can be removed. In Applications, make sure to change the tense into third person. Limitations seems a little vague and can be expanded and reworded so run-on sentences are not used. Erin R (Chem 455/505 Group 1) 17:00, 22 October 2016 (UTC) — Preceding unsigned comment added by Goblue2017 (talk • contribs)

Additional comments from the librarians

The whole second half of the Applications section sounds a lot like it was copied and pasted from a primary research article that used the technique: its use of "us" and references to "this experiment" are nonsensical in the encyclopedia context. I strongly suggest rewriting this so that it sounds more like a paraphrase and less like plagiarism. I also recommend more aggressively linking your technical terms to their existing Wikipedia articles. ScottMLibrary (talk) 15:54, 24 October 2016 (UTC)

Responses to the peer reviews

• many hyperlinks were added to the page to allow readers to clarify jargon
• the "methodology" section was organized to separate freely-diffusing and surface-immobilized systems
• citations were added to the section that spoke about confocal microscopy
• the advantages of a three color system were added to the "advantages" section
• first person was changed to third person throughout the article
• expanded on applications to protein-protein and protein-nucleic acid experiments using smFRET.
• Reworded second half of application section.
• reworded limitation section.

Magnus Ma (talk) 19:17, 17 November 2016 (UTC)

— Preceding unsigned comment added by Magnus Ma (talk • contribs) 00:45, 16 November 2016 (UTC) 

Heavy overemphasis on data analysis

Almost half the content of the article is devoted to data analysis, and only of TIRF, with nothing to say for confocal analysis which differs by quite a lot. As well as being specific to TIRF it is also regularly specific to just one analysis package (postFRET), which happens to be the package made by the user who wrote this section, despite the fact that there are many other analysis packages which are just as widely used and may work differently. This section could do with being cut down significantly, and being far more general in it's description, as it is currently misleadingly unrepresentative of the technique because of its focus. I would recommend modelling it after the article on single particle cryo-EM (https://en.wikipedia.org/wiki/Single_particle_analysis) as it is a comparable biophysical technique and succeeds in being succinct and generalizable. More detail on confocal would also help balance the article.

FluorescentAdolescence (talk) 18:30, 27 April 2020 (UTC)