Accomplishments of “Old-Fashioned” Electron Microscopy in the Period of Dominance of Immunofluorescent Methods

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a well-composed review of the author’s work in the field of electron microscopy, clearly demonstrating the unique opportunities provided by this method to interrogate a wide range of tissue- and cell-level phenotypes. The primary goal of this review is to highlight the power of transmission electron microscopy for analyzing biological specimens and resolving complex data interpretation challenges. The manuscript will be of interest to a broad community of cell biologists.

A small number of editorial issues should be addressed prior to publication. These are largely minor and do not detract from the overall quality of the work.  Specific comments are outlined below.

1. Line 68 – Please close the open parenthesis.

2. Figure 1 legend vs. main text – “Wild-type” is used in the figure legend, whereas “wild type” is used in the main text. Please ensure consistent usage throughout the manuscript.

3. Mitochondrial phenotypes – Because Type I and Type II mitochondria are central to this study, the description of these phenotypes should be introduced earlier in the manuscript.

4. Line 101 – Please change “electron microscopic analyses” to “electron microscopy analyses.”

5. Line 107 – Please introduce and define the abbreviation SLP2 at first use (this is applicable to all abbreviations in the manuscript).

6. Line 162 – Consider revising the title to:
   “What CB1R-L31 Immunolabeling Reveals About Mitochondrial Function.”

7. Line 166 – Please close the open parenthesis.

8. Line 195 – Please define DAB-Ni at first mention.

9. Figure 9 – Please replace “Destroy of organelles” with “Organelle destruction” or “Organelle degradation.”

Comments on the Quality of English Language

Needs to be checked throughout

Author Response

Reviewer 1

Comments and Suggestions for Authors

This is a well-composed review of the author’s work in the field of electron microscopy, clearly demonstrating the unique opportunities provided by this method to interrogate a wide range of tissue- and cell-level phenotypes. The primary goal of this review is to highlight the power of transmission electron microscopy for analyzing biological specimens and resolving complex data interpretation challenges. The manuscript will be of interest to a broad community of cell biologists.

A small number of editorial issues should be addressed prior to publication. These are largely minor and do not detract from the overall quality of the work.  Specific comments are outlined below.

1. Line 68 – Please close the open parenthesis.

Corrected.

1. Figure 1 legend vs. main text – “Wild-type” is used in the figure legend, whereas “wild type” is used in the main text. Please ensure consistent usage throughout the manuscript.

Corrected in two lines in the figure legend.

1. Mitochondrial phenotypes – Because Type I and Type II mitochondria are central to this study, the description of these phenotypes should be introduced earlier in the manuscript.

Corrected. We inserted the following sentences in Charter 2 (lines 65-70):

“… we detected binding of anti-cannabinoid type 1 receptor (CB₁R) sera with mitochondria of two morpho-functional types whereas many other mitochondria stay immunonegative (Figure 1A-F and [6]). One type of immunopositive mitochondria, designated as ‘type 1’, contained immunoreaction end-products on the outer membrane and inside the cristae. The other type, designated ‘type 2’, showed immunolabeling in the mitochondrial matrix. (See next chapters for discussion of probable functionality of these mitochondrial types.)”

1. Line 101 – Please change “electron microscopic analyses” to “electron microscopy analyses.”

Corrected.

1. Line 107 – Please introduce and define the abbreviation SLP2 at first use (this is applicable to all abbreviations in the manuscript).

Corrected.

SLP2 is defined as stomatin-like protein 2 at line 87;

DAB-Ni is defined as nickel-intensified 3,3′-diaminobenzidine-4HCl at line 98 (legend to Figure 1) and line 118 in the main text;

TUNEL is defined as terminal deoxynucleotidyl transferase dUTP nick end labeling at line 262.

We also checked the following abbreviations:

three-dimensional (3D) is defined at line 49;

the last 31 amino acids of the C-terminus (L31) is defined at line 124;

Golgi apparatus (GA) is defined at line 299;

nuclear envelope (NE) is defined at line 480;

piercing nuclear hernia’ (PNH) is defined at line 486;

regulated cell death (RCD) is defined at line 508;

neural progenitor cells (NPCs) is defined at line 582.

1. Line 162 – Consider revising the title to:
   “What CB1R-L31 Immunolabeling Reveals About Mitochondrial Function.”

Corrected. We are grateful for this suggestion.

1. Line 166 – Please close the open parenthesis.

Corrected.

1. Line 195 – Please define DAB-Ni at first mention.

Corrected. See above.

1. Figure 9 – Please replace “Destroy of organelles” with “Organelle destruction” or “Organelle degradation.”

Corrected. We replaced it with “Organelle degradation”. Thank you.

Comments on the Quality of English Language

Needs to be checked throughout

Proof reading was done by a native American English-speaking person.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is good written and has a logical structure but requires some improvement. The authors cite themselves but the paper looks interesting for readers in its present form. The current version can be improved by the following way.

1) The manuscript is focused on benefits of transmission electron miscoscopy in comparison to fluorescent microscopy. At the same time, the Conclusions are not related to both Abstract and Introduction sections. 

Also, the Conclusions should be rewritten in a different way, more like a discussion than like short notes based on previous publications cited in the paper.

2) A general concept scheme of the manuscript is required to make its topic, results and conclusions more clear for readers.

3) Why the authors choose the current list of bioobject for investigation? What are the limitations of TEM and benefits of fluorescent microscopy?

Author Response

Reviewer 2

Comments and Suggestions for Authors

The manuscript is good written and has a logical structure but requires some improvement. The authors cite themselves but the paper looks interesting for readers in its present form. The current version can be improved by the following way.

1) The manuscript is focused on benefits of transmission electron microscopy in comparison to fluorescent microscopy. At the same time, the Conclusions are not related to both Abstract and Introduction sections. 

We are grateful for this recommendation. We made following corrections in the Abstract:

“We are deliberating the following topics: interpretation of unusual results of immunolabeling; a novel method for in situ identification of cells undergoing mitochondrial disorder and necrosis-like death; the sequence of organelles’ reorganization in dying cells; simultaneous rupture of nuclear and plasma membranes in migrating neurons; role of centrosomes in lateral expansion of cerebral cortex.”

and in Introduction:

“Particularly, , we consider the following aspects: (1) double selectivity of antibodies producing unusual immunolabeling of mitochondria that helps for in situ identification of cells undergoing mitochondrial disorder and necrosis-like cell death; (2) the sequence of morpho-functional reorganization of mitochondria and Golgi apparatus in anoxia-exposed mouse embryo brain; (3) novel mechanism of cell pathology identified through simultaneous rupture of nuclear and plasma membranes in neurons migrating through tightly packed brain tissue; and (4) the role of centrosomes - the main cytoskeleton organizing center - in prolonged lateral expansion of rhesus macaque’s cerebral cortex.”

Also, the Conclusions should be rewritten in a different way, more like a discussion than like short notes based on previous publications cited in the paper.

According to the Instruction for authors of IJMS, the chapter Conclusions “… can be added to the manuscript if the discussion is unusually long or complex”. This is the case of our article that contains long discussions through the text. We prefer to keep Conclusions as is, but can remove this chapter, if the Editors require this.

2) A general concept scheme of the manuscript is required to make its topic, results and conclusions more clear for readers.

We made several corrections through the text. See our replays to other comments.

3) Why the authors choose the current list of bioobject for investigation?

Corrected. We are grateful for this suggestion. We added the following sentences in the first paragraph of Chapter 7:

“We investigated if CB₁R-L31 serum can be used for identification of disordered mitochondria in other tissues and organs. We chose mouse liver and white adipocytes because these cells are morpho-functionally distinct from brain cells.”

We also added the following sentence at the end of Chapter 7:

“Overall, our application of CB₁R-L31 serum for brain, liver and adipose tissue indicates that immunohistochemical detection of SLP2-like immunopositive type 2 mitochondria can serve as a marker for identification of necrosis-like ultrastructural pathology and cell death in different tissues and organs.”

What are the limitations of TEM and benefits of fluorescent microscopy?

Corrected. We recognize the limitation of transmission electron microscopy as a complicated method and the benefit of fluorescent microscopy as a relatively simple method.

We highlighted it in the abstract as follows:

” Although procedures for electron microscopy are in general more elaborate (particularly for simultaneous immunolabeling of multiple antigens) compared to fluorescent microscopy ...”

We also added the following sentence in Introduction:

“As an advantage of immunofluorescent microscopy, we must highlight relative simplicity to perform multiple antigen labeling (triple and more), whereas double labeling for transmission electron microscopy is much more elaborate.”

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, thank you so much for the corrections,  now the paper is  better structured. Please, include some discussion to the Conclusions section. Now the abstract and the conclusions do not correlate well one to another. Also the conclusions are too short and do not discuss perspectives of the topic (please, see the title).

Author Response

We are grateful that you insisted on rewriting the chapter Conclusions. Your recommendation was really helpful.

We considerably increased this chapter incorporating elements of discussion in it. See added text in red font.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is focused on the benefits of the "old fashioned" electron microscopy in comparison with modern fluorescent techniques. Now all sections of the text is significantly improved and I guess the manuscript could be accepted in its present form.