edits to abstract to improve flow

00_index.md

@@ -9,12 +9,12 @@ numbering:
 
 Static figures are the standard interface for scientific discourse and peer review, so microscopists typically communicate experimental results through representative images and plots.
 This article focuses on virtual microscopy, a novel visualization strategy for researchers to share microscopy data in publications.
-Web-based microscopy maps improve the transparency, accessibility, and archival of bioimages as an unbiased alternative to representative images.
-These features make virtual microscopy useful for the critical appraisal of observational studies.
-Using this approach, an original claim of reovirus-induced endoplasmic reticulum remodeling was reexamined.
+Web-based microscopy maps improve the transparency, accessibility, and archival of bioimages as an unbiased alternative to representative images. 
+With the use of purpose-built image display components, the benefits of virtual microscopy can be realized without sacrificing the narrative advantages of traditional representative figures. 
+To show how this approach to data visualization facilitates the critical appraisal of observational studies, an original claim of reovirus-induced endoplasmic reticulum remodeling was reexamined.
 Virtual microscopy subjects the results of qualitative experiments to unbiased peer review by giving readers direct access to the data.
 While data visualization can be refined to minimize subjective biases, the interpretation of data inevitably reflects the author's perspective.
-Virtual microscopy allows readers to interpret the same data as the author, which is a meaningful step forward, yet how authors determine the outcome of qualitative microscopy experiments remains unclear.
+Virtual microscopy allows readers to interpret the same data as the author and independently assess the results.
 
 +++
 

01_introduction.md

@@ -4,27 +4,27 @@ numbering:
   enumerator: 0.%s
 ---
 
-Representative microscopy images are visual communication tools used by microscopists to present research results to other scientists.
+Representative microscopy images are visual communication tools that microscopists use to present research results to other scientists.
 The earliest representative microscopy images were hand-drawn in Robert Hooke's [Micrographia](<wiki:Micrographia>) in 1665.
 Today, roughly three-quarters of publications in biomedical journals report at least one microscopy image [@doi:10.7554/eLife.55133].
-There is an unspoken expectation that scientists rely on these images to be honest and accurate.
+Readers expect these images to be honest and accurate.
 However, microscopy image selection and quantitative analyses thereof are vulnerable to bias and deception [@doi:10.1242/jcs.261567].
-Besides cherry-picking, scientists can sincerely misinterpret observations.
-The problem is that readers find it difficult to critically evaluate representative images.
-Furthermore, archival and review of primary microscopy data is disorganized and time-consuming, if at all available.
-These problems can be addressed with novel visualization strategies like [virtual microscopy](<wiki:Virtual_microscopy>) that transmit entire interactive datasets over the internet.
-An example was the unbiased presentation of electron microscopy data to readers as interactive digital maps — or microscopy maps [@doi:10.1083/jcb.201201140].
+Besides cherry-picking, scientists can sincerely misinterpret observations. 
+Although narratively useful, representative images inherently discard data, precluding independent analysis of results by readers.
+Furthermore, archiving and review of primary microscopy data are disorganized and time-consuming, if available at all.
+These problems can be addressed with novel visualization strategies such as [virtual microscopy](<wiki:Virtual_microscopy>), which transmit entire interactive datasets over the internet.
+An example is the unbiased presentation of electron microscopy data to readers as interactive digital maps — or microscopy maps [@doi:10.1083/jcb.201201140].
 In practice, microscopy maps are easy to share but are accessed via external links [@doi:10.1242/jcs.262198].
 
 [Reproducibility](<wiki:Reproducibility>) is an essential principle of the [scientific method](<wiki:Scientific_method>) for acquiring knowledge, yet there is a contentious [reproducibility crisis](<wiki:Replication_crisis>) in science.
 From the perspective of biomedical researchers, the perceived cause is the pressure to publish [@doi:10.1371/journal.pbio.3002870].
 Repeatability within the same study by the same researchers is distinguished from replicability by independent researchers using the same methodology.
 Large-scale replicability studies found that only 11% [@doi:10.1038/483531a] and 25% [@doi:10.1038/nrd3439-c1] of key findings from preclinical cancer research papers were replicable.
-The replication crisis is multifaceted, but the irreproducibility of representative images deserves scrutiny in its own right.
+The replication crisis is multifaceted, but the irreproducibility of representative images deserves scrutiny.
 According to one study, replication rates for representative images ranged from 27% to 90% [@doi:10.7554/eLife.71601].
 This wide range reflects how difficult it is to assess the replicability of representative images without dedicated tools.
 A feature of virtual microscopy is the unbiased sampling and transmission of multi-scale image data, making it useful as a method to replicate representative images. 
 If results are reproducible, original claims should be readily observable in microscopy maps, which capture a broader range of variation.
 Here, microscopy maps were used to reassess the claim that mammalian orthoreovirus remodels the endoplasmic reticulum (ER) of cells during infection [@doi:10.1128/mBio.01253-18].
-Using the protocols described in the original paper, hundreds of overlapping tiles were sampled at high-magnification from replicate immunofluorescence experiments on a fully motorized confocal microscope.
+Using the protocols described in the original paper, hundreds of overlapping tiles were sampled at high magnification from replicate immunofluorescence experiments on a fully motorized confocal microscope.
 Microscopy maps were embedded in the article using the next generation file format OME-Zarr [@doi:10.1007/s00418-023-02209-1] and custom viewers based on Viv [@doi:10.1038/s41592-022-01482-7].

02_approach.md

@@ -5,8 +5,8 @@ numbering:
 ---
 
 Already adopted by some clinicians and educators, virtual microscopy could also benefit researchers as an interface for communicating and interpreting image data.
-Indeed, interactive microscopy maps were first proposed for precisely this purpose, as an unbiased way to present electron microscopy data [@doi:10.1083/jcb.201201140].
-Free and open-source solutions [@doi:10.1242/jcs.262198] and recent advances in file formats [@doi:10.1007/s00418-023-02209-1] now make this feasible, yet these tools have not been adopted in scientific publishing.
+Indeed, interactive microscopy maps were first proposed for this purpose — as an unbiased way to present electron microscopy data [@doi:10.1083/jcb.201201140].
+Free and open-source solutions [@doi:10.1242/jcs.262198] and recent advances in file formats [@doi:10.1007/s00418-023-02209-1] now make this feasible, yet these tools have not been used in scientific publishing.
 Our approach combined automated image acquisition, multi-scale [pyramid](<wiki:Pyramid_(image_processing)>) formats and embedded interactive viewers ([](#abstract)).
 
 ```{figure} ./figures/Virtual_microscopy.png
@@ -15,11 +15,11 @@ Virtual microscopy. 1. Automated dataset collection with any suitable microscope
 ```
 
 Elemental Microscopy supports two interactive viewers; [Vizarr](https://github.com/hms-dbmi/vizarr) [@doi:10.1038/s41592-022-01482-7] and [OpenSeadragon](https://openseadragon.github.io/), enabling two methods of virtual microscopy based on OME-Zarr or Deep Zoom. 
-Authors can also build custom viewers. 
+Authors can also build custom viewers and embed them within online articles.
 Brodrick developed [microATLAS](https://github.com/LadInTheLab/microATLAS-widget), which was used to view OME-Zarr data in this article. 
 All three viewers are readily embeddable in Elemental Microscopy. 
 For Vizarr and OpenSeadragon, authors simply copy the markdown and replace the source URL with a link to their own data. 
-For microATLAS, authors are encouraged to use the [widget builder](https://ladinthelab.github.io/microATLAS-widget/builder.html) to generate code.
+For microATLAS, authors are encouraged to use the [widget builder](https://ladinthelab.github.io/microATLAS-widget/builder.html) to generate embedding code that specifies both the image and relevant customizations.
 
 ## OME-Zarr
 
@@ -90,7 +90,7 @@ By default, readers cannot control dimensions like channels or time, so it is be
 ```
 
 :::{dropdown} Imaging
-Data collection was fully-motorized for unbiased sampling of large panoramas from replicate immunofluorescence experiments.
+Data collection was fully motorized for unbiased sampling of large panoramas from replicate immunofluorescence experiments.
 Grids of overlapping tiles were acquired with a robotic XY stage (IX3-SSU) and Z-drift compensation (IX3-ZDC).
 Coverslips were imaged with an Olympus Fluoview FV3000 confocal microscope using a 100X objective lens (Olympus model UPLAPO100X, NA 1.50) in immersion oil. 
 Pinholes in each channel were maximized to capture the widest depth of field.

04_plasmids.md

@@ -14,7 +14,7 @@ An earlier experiment combining moxGFP::μNS with these constructs was inconclus
 
 Cells transiently expressing mCherry-ER fusions were fixed 24 hours after transfection with TransIT-2020 and Lipofectamine 3000.
 Parker identified saturated signal in the original experiment and advised Barkley to repeat it, adjusting illumination settings individually.  
-The corrected maps are presented below (Figure 3.1) and the [saturated maps](#saturated_page) (Figure 15.1) and [discarded maps](#discarded_page) (Figures 16.1 to 16.5) are presented in the Supplemental.
+The corrected maps are presented below (Figure 3.1), and the [saturated maps](#saturated_page) (Figure 15.1) and [discarded maps](#discarded_page) (Figures 16.1 to 16.5) are presented in the Supplemental.
 
 :::::{figure}
 :label: figure3

08_results.md

@@ -5038,6 +5038,6 @@ Calreticulin and reovirus μNS antigens were detected by immunofluorescence.
 Chicken antiserum was paired with AlexaFluor647 secondary antibodies. 
 Nuclei were labelled for 15 minutes in Nucblue Fixed Cell Stain ReadyProbes reagent (containing DAPI) (R37606, Fisher) diluted in PBS, then a 25×25 grid of 625 fields was imaged. 
 Barkley annotated transfected cells expressing mCherry-KDEL based on the presence or absence of reovirus infection using the microATLAS builder. 
-Transfected-infected cells were labelled "Coincidence" because the co-occurrence of transfection and infection in the same cell is incidental, while transfected-uninfected cells were labelled "Transfection".
+Transfected-infected cells were labelled "Infected", while transfected-uninfected cells were labelled "Uninfected".
 Ambiguous cells were left unlabelled, and it is likely that some transfected cells were overlooked.
 :::

09_discussion.md

@@ -6,24 +6,23 @@ numbering:
 
 Every academic discipline has its own ways of deciding what counts as valid knowledge (see [epistemology](<wiki:Epistemology>)).
 If biomedical researchers endorse empiricism and objectivity, then why are representative images sufficient to make claims?
-A representative image shows that an observation is possible, but a single image cannot describe frequency or variance.
+A representative image shows that an observation is possible, but a single image cannot capture frequency or variance.
 The scientific method demands reproducibility, yet representative images offer no way to communicate repeatability without quantitative analysis.
-In practice, representative images can show rare, idealized, or exaggerated examples to embellish results.
-The literature is full of exciting outcomes, but at the bench, experiments are nuanced and prone to failure.
+In practice, representative images often show idealized or exaggerated examples to best illustrate the authors' interpretations, but these may not capture the variability found in the data.
+The literature is full of exciting outcomes, but at the bench, experimental results are nuanced.
 
 Representative images are figures, which should not be confused with data.
 Data is the unprocessed file from the microscope, while figures are visual representations of data, shaped by the microscopist.
-Figures are the interface for peer review between the experimentalist and coauthors, principal investigators, reviewers and readers.
-Indeed, peer review seldom involves data and scientific discourse is limited by the information provided in figures.
+Figures are the interface for peer review between the experimentalist and coauthors, principal investigators, reviewers, and readers.
 This bottleneck is alleviated by virtual microscopy, which is practical to the broader bioimaging community.
-For example, OME-Zarr [@doi:10.1007/s00418-023-02209-1] supports several modalities including multiplexed imaging, spatial transcriptomics, light-sheet, infrared, scanning EM and digital pathology.
+For example, OME-Zarr [@doi:10.1007/s00418-023-02209-1] supports several modalities including multiplexed imaging, spatial transcriptomics, light-sheet, infrared, scanning EM and digital pathology, and its pyramidal, tiled structure ensures that even extremely large images remain responsive during interactive analysis.
 To maximize accessibility for readers, embedded interactive viewers retrieve data from cloud storage, which is supported by this journal.
 Virtual microscopy requires an internet connection, but this limitation is relatively unimportant for contemporary and future publishing.
 Web-based microscopy maps are trivial to make and share on Github [@doi:10.1242/jcs.262198], so they can be readily requested by reviewers of all journals.
 Data transparency could incentivize publishers to adopt this technology as an alternative to static figures.
 Because it nurtures open and rigorous peer review, microscopists and principal investigators might find virtual microscopy advantageous to make more robust claims with their bioimaging data.
 
-Barkley advocated for the use of virtual microscopy as an "unbiased" method to replicate highly magnified representative images.
+We advocate for the use of virtual microscopy as an "unbiased" method to replicate highly magnified representative images.
 This process involves automated sampling of reasonable populations from which the original claim should be reliably observed.
 Virtual microscopy is "unbiased" to the extent that the data was sampled with robotics and is hosted interactively on the web.
 To test this approach, an immunofluorescence imaging experiment in reovirus-infected cells [@doi:10.1128/mBio.01253-18] was reexamined with virtual microscopy.

myst.yml

@@ -56,7 +56,6 @@ project:
         - Software
         - Visualization
         - Writing - original draft
-        - Writing - review & editing
     - id: ajbrodrick
       name: Andrew James Brodrick
       email: ajb64@umd.edu
@@ -72,6 +71,7 @@ project:
         - Methodology
         - Software
         - Visualization
+        - Writing - review & editing
     - id: jslparker
       name: John Stuart Leslie Parker
       email: jsp7@cornell.edu