- Aug 2024
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www.biorxiv.org www.biorxiv.org
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first 10 Principal Components (PCs)
How many PCs can explain 95% of the variance? And how many features went into the PCA?
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host immunity is modulated to facilitate endosymbiosis
It would be useful to show a schematic of the differences in aposymbiotic and symbiotic stony corals, kind of like a graphical abstract. The abstract was hard to follow without reading the details of the study, and it would be nice to see the overall findings in a visual way. This could get across the idea that neither the bacterial communities nor the proportion of immune cells differ between symbiotic states, but the gastrodermis I cells downregulate immune genes when algae is in close contact. That begs the question, will you follow up to determine what chemical cues from the algae are sensed by the gastrodermis cells to alter their gene expression?
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Immune Cell
I don't think it's necessary to capitalize immune cell while naming the type of cluster.
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aposymbiotic branches were transferred back to common garden aquaria and maintained for at least 2 months of recovery prior to physiological and multiomic profiling
How do these corals acquire their symbionts? Is it surprising that with 2 months of recovery following the bleaching even that the symbiont load remained so low? Or is there a sensitive window that has closed for corals at this stage of development?
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- Jul 2024
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www.biorxiv.org www.biorxiv.org
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It serves as a proof of concept for a high throughput, remote technique applicable to field conditions.
This seems great for surveying plant physiology for ecological field work. I didn't see anywhere an estimation of the throughput though, can any numbers be assigned to this to help a researcher see how the technique would increase teh efficiency of their work?
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especially NDVI (Fig. 5 A, Fig. S5 A), SR (Fig. 5 B, Fig. S5 B), RENDVI (Fig. 5 F, Fig. S5 F), mRENDEVI (Fig. 5 G, Fig. S5 G), mRESR (Fig. 5 H, Fig. S5 H0, VOG1 (Fig. 5 I, Fig. S5 I), PRI (Fig. 5 L, Fig. S5 L), SIPI (Fig. 5 M, Fig. S5 M), RGRI (Fig. 5 N, Fig. S5 N), PSRI (Fig. 5 O, Fig. S5 O) CAR1 (Fig. 5 P, Fig. S5 P), and CAR2 (Fig. 5 Q, Fig. S5 Q).
Can you spell out these acronyms here so the reader can refer to this section when interpreting the y-axes on Fig. 5 and 6? I see that they're listed in the abbreviations, but it would handy to have them written out in this section of the results.
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Welch t-test.
It would be worth reporting the sample sizes for each treatment, as that would help the reader understand why you chose different tests (like Welch's t-test). Also for D, I am assuming you actually performed an ANOVA and then a Tukey's post-hoc test, so you should report the ANOVA statistics (F value, df, p value).
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Fig. 4
I see now that the label for Darkened should really be "Dark-adapted", is that true?
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Fig. 2.
Same comment as for Fig. 1 but also it would help to make the label "Dark" more parallel. Is that a "No light" treatment as control? Consider new labels to make it clearer as to whether Dark means a condition or an observation as in Fig 1, "Darkened" seems like an observation.
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may be observed with the naked eye
Interesting, what is observed by eye? Is it a darkening of a green hue?
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ig. 1.
small note - but consider labeling the panels "low" and "high" blue light and keep the info on the values for each treatment in the caption. This would improve readability.
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- Jun 2024
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www.biorxiv.org www.biorxiv.org
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P values from Scheffe post-ANOVA show no significant difference between blebbistatin and Y27632 treatments either parallel or perpendicular to the edge and no significant difference between the conditions parallel to the edge
Was the F statistic from the ANOVA significant and were the comparisons actually between drug treatment and the control? If so, I would think a Tukey's post-hoc test would be more appropriate. If the ANOVA was not significant, then why are none of the p values from the Sheffe post-hoc test not significant? I was suprised because it looks like in panel d that the control and treatments look different.
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In summary, our findings reconceptualize the front of the cell as a dynamic, ‘membrane-less’ organelle 35, which perpetually directs polymerizable proteins to precisely where they are most needed
Wow, this is a really important finding that reshapes how we think about cells can efficiently and rapidly alter their architecture in response to environmental changes.
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- May 2024
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www.biorxiv.org www.biorxiv.org
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Results
This seems like an extensive study, but the figures have so many panels and the captions are so long, that it make it hard for the reader to quickly understand the key points. It would greatly benefit the reader if you streamline the figures and reduce redundancy within the captions (across letters in the panels and also with methods sections). Because of the different time courses, perhaps a schematic showing the overall experimental design in the first panel woud be useful for some figures.
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our study reveals an autophagy-regulated metabolic rheostat that gauges cellular integrity during viral infection and degrades cell death executors to avoid catastrophic amplification of immune signaling.
Is there a more direct way to state the findings?
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EDS1
Just a note for readers outside of the Arabidopsis field that EDS1 hasn't been defined yet in the abstract.
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www.biorxiv.org www.biorxiv.org
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By tuning the network depth, wewere able to restrict the gradients only to the most important differences among classes. InEvoCellNet, the final classification is precisely overlaid with cytoplasmic features.
This is an interesting approach to ensure the classifier picks up on cytoplasmic features to better differentiate cell stages. You may be interested in this pub, which uses a ResNet model on brightfield timelapses of nematodes. It would be interesting to see how the brightfield data (available in the GitHub) perform with the four models explored in your study given potential differences with intracellular hallmarks that could be more apparent by DIC.
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- Apr 2024
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www.biorxiv.org www.biorxiv.org
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the study affirms the potency of SWCNT-based spectral fingerprinting, particularly when coupled with machine learning, as an invaluable tool for precisely categorizing cellular states based on complex spectral data.
This is an interesting application of machine learning to expand the phenotyping capacity of cell types that are hard to distinguish without more intensive or destructive methods.
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In a previous study, we have shown that DNA-SWCNTs can be used to map intracellular processes based on modulations in their Raman spectra.
Do you suspect there are any intrinsic differences in the Raman or NIR spectral for M1 or M2 macrophages without the addition of the DNA-SWCNTs? It would be interesting to know to what extent this works as a label-free approach for applications without the addition of single-walled carbon nanotubes.
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- Mar 2024
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www.biorxiv.org www.biorxiv.org
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Figure 2.
I found panels A-C confusing and have questions that may help you clarify to the reader. Is the grayscale image to the right of the four panels a merge of the 4 markers shown in C? If so, I might write merge on it. I was trying to connect the panels in C to the red line in A because I thought it was an example of how you might classify CD4+ T cells based on the high signal for CD3, CD 45, CD4, and CD8. But then I saw the image to the right of them. Regardless, I would switch the top two panels in C so that they read in the order of the markers in A and B (CD3 and then CD45).
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In the 43-plex kidney images, pSAM detected a wide range of cell classes with highly variable prevalences, suggesting that it can accurately detect rare cell types.
This tool seems quite robust for classifying many cell types based on multiple markers. I'm wondering if you see a useful application of the tool as identifying unusual combinations of markers that deviate from the training data?
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- Feb 2024
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www.biorxiv.org www.biorxiv.org
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Together, the need for increased oxygen supply due to tissue expansion and the concurrent reduction in pulmonary vasculature will lead to a quicker deterioration of lung and vascular function overall.
I really like the illustration of what this study found in the graphical abstract. I don't see where the schematic incorporates the hypoxia component. If that is relevant then it may be worth making that clearer.
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during the resolution phase
It would be helpful to the reader to hear more about how the resolution phase was determined.
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Together these parameters provide an accurate representation of the lung vascular architecture and its impact on inflammation.
It would be interesting to determine which of the 6 different parameters explained most of the variation in lung architecture in case it only takes a couple measurements to assess response to inflammation.
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We utilised fluorescent avidin staining to analyse in detail the profile of degranulated CTMCs as we and others have demonstrated that it provides an accurate measurement of the localisation and cellular interactions of extracellular CTMC granules
I'm interested in whether you saw a difference in staining between granules that were extracellular to the MCs or the ones associated with changes in pericyte morphology.
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- Jan 2024
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www.biorxiv.org www.biorxiv.org
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Methods
Very much appreciate the level of detail for the choices for each step of the methods. This makes understanding the adjustable parameters and functions of RamanSPy much more approachable.
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Pipeline II was deemed the most robust,
It would be helpful to hear more about how the level of robustness was determined.
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- Dec 2023
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www.biorxiv.org www.biorxiv.org
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Further, prior work shows that orientation-invariant encoders have better performance for supervised models on biological and medical data45, 46, 47, despite supervised learning having more techniques to enforce invariance (e.g. data augmentation). There are also theoretical arguments supporting this idea: a central idea in geometric deep learning is that enforcing known data invariances in neural networks should improve representations
I'm curious about the authors' opinion about whether a pattern needs to be apparent to the human eye in order to be detected by a computer. These statements suggest that the supervised models perform worse than autoencoders like the one described here.
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An object is ‘in contact’ with another object if its separation is smaller than 2 pixels.
I'm curious whether the authors considered using the model to identify specific organelle contacts of interest to further investigate using deconvolved images and confirming the contact.
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mitohconria
typo, mitochondria
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images
typo, should be imaged
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15,000 images were generated for each condition
This is useful to know. Do you have a sense that 15,000 images was near the minimum number needed or whether it was overkill for the training?
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Moving beyond segmentation-only data, we take grayscale nuclei images undergoing mitosis24, and show that modelling texture improves the morphologic profiles
Texture is something that can be harder to quantify than cell shape so it is great that it can be captured by models like this one since it improves the morphologic profiles.
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- Nov 2023
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www.biorxiv.org www.biorxiv.org
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most of the signal presumably occupying the stroma of the chloroplast. A greater accumulation of mNG fluorescence was observed in the center of chloroplast lumen, with the highest green signal presumably located in the zone where pyrenoid is located (Figure 5a, progressions 3-4). Notably, most of the mNG fluorescence signal does not co-localize with chloroplast fluorescence.
It is interesting to see the stratification of chloroplast signal between the mNG fluorescence and chloroplast autofluorescence. Is the bulk of the nucleus within the empty space of the chloroplast autofluorescence? What accounts for the DAPI staining throughout the cell body?
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Other works have described that exclusion of proteins higher than ∼50kDa from the pyrenoid matrix may occur [Mackinder et al., 2018], this might be happening with mNG due its effective molecular weight observed at ∼70kDa by native-PAGE in this work.
Would it be possible to synthesize this reporter with a lower MW to explore the heterogenous distribution further?
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www.biorxiv.org www.biorxiv.org
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However, the intestinal loads of the evolved phages were decreasing faster than the ancestor (Figure 4C). This correlated with the rapid appearance of resistant mutants in the S.Tm* population exposed to evolved phages (Figure 4D and S7), demonstrating that phages able to kill S.Tm despite phase variation favor genetically resistant mutants, which, in turn, impairs the replication of the phages in the infected mice.
Given that you identified two evolved phages within a mouse, I'm interested in your speculation as to how frequently you think this occurs in nature. Do you think that the two phages occupy the same intestinal niche or is there evidence that they are stratified along the GI tract?
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www.biorxiv.org www.biorxiv.org
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In complex microbial communities, all metabolically active cells will incorporate deuterium (D) from D2O into their biomass during synthesis of new macromolecules34. The newly formed carbon-deuterium (C-D) bonds can then be used as a read-out of microbial activity. Detection and quantification of C-D levels in single microbial cells can be achieved using SRS, a method that efficiently excites the Raman active vibrational modes coherently with two synchronized ultrafast lasers
This seems like a comprehensive and sensitive readout of microbial activity. It might be worth defining SRS as Stimulated Raman Scattering as the reader could mistake it for Spontaneous Raman Scattering.
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As ENT-Hi samples showed a strong, Raman unspecific signal during SRS pump-probe detection, we explored the origin of this signal and concluded that this is a photothermal (PT) signal originating from entacapone bioaccumulation within microbial cells (Supporting Information Text).
I'm curious if you can share insight into how to design an SRS imaging experiment to allow for this type of unexpected observation of the time-dependent photothermal signal.
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However, many of the detected effects were drug-specific, with ENT-Hi decreasing and LOX-Hi increasing total abundances of the genera Anaerostipes, Fusicatenibacter, Ruminococcus torques group, Eubacterium hallii group, Erysipelotrichaceae group UG-003 and Roseburia.
These opposite effects on the same group of strains by each drug is quite interesting. I'm curious about your interpretation as to why the ENT-Hi community looks so similar to the inoculum at 6 h except for the increase in E. coli abundance. One might think there's no effect of ENT-Hi on the community, but something is changing relative to the inoculum to allow E. coli to grow.
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Major shifts in the microbial community composition, as determined by 16S rRNA gene amplicon sequencing analyses, were detected in response to ENT-Hi, LOX-Hi and LOX-Low treatments
Is the major shift for the LOX-Hi and LOX-Low treatments the reduction in Bacteroides that is present at 6h but stronger at 24h? If so, it might be useful to call that out specifically for the reader because the fact that Bacteroides is affected might catch their attention.
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- Sep 2023
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arxiv.org arxiv.org
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Alternatives like unsuper-vised learning provide flexibility but may compromise accu-racy.
Perhaps consider commenting on the differences between supervised, semi-supervised, and unsupervised learning and which you suggest as optimal to reduce photoxicity.
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Drawing inspirationfrom the delineation provided in (22), we distinguish strate-gies that aim either to surmount the physical limitationsintrinsic to live fluorescence microscopy imaging (i.e.,acquisition speed or illumination) or to enhance the contentin less qualitatively superior but more sample-friendly imagedata.
Consider revising into shorter and more declarative sentences.
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Given this, alongside time-consuming data an-notation processes, has
Revise this to ensure a proper subject.
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Deep learning modelscan be used to run microscopy acquisitions that use lower fluorescence light intensities or illuminate the sample less often. For this, after the imaging experiment,
It sounded like the topic was using D-L models to iterate during acquisition but this refers to applying the model after the imaging experiment (and data are acquired), so please clarify.
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CLEM
define CLEM in this section
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confocal mi-croscop
see point about Fig. 2B
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Yet
Eliminate and start with "An"
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less commonly employed
"rarer"
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b) The effect ofdifferent light microscopy modalities
Consider clarifying the type of confocal referred to here and including both spinning disk and point-scanning confocal methods to this graph.
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Despite challenges associated with varyingspecimen resistances to light damage, establishing universalquantitative benchmarks across diverse samples could har-monise these effects and enhance reproducibility in the imag-ing context.
This is a great point, consider condensing this section to get to this point across sooner.
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However,despite the interrelation between photobleaching and photo-toxicity (20), these phenomena exhibit distinct features andcan occur independently.
Consider flipping this sentence to first state that these two phenomena can occur independently but are related.
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While helpful and versatile, fluorescence microscopy illumi-nation entails an additional source of ROS formation
Consider revising this topic sentence to include photobleaching and eliminate redundancy with the previous paragraph.
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environment’
typo, eliminate the '
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Moreover, molecules naturally presentin cells (Table 1) undergo degradation via exposure to light-induced oxidative stress, canonically produced during the flu-orescence excitation process
Consider saying, "Many biomolecules undergo degradation when exposed to specific oxygen radicals (Table 1)."
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The literature exten-sively documents the impacts of fluorescence excitation light,especially UV light on DNA,
This point is clear through the citations so consider sticking to the point of UV light damaging DNA.
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Light irradiation to excite fluorescence, in addition to induc-ing oxidative stress by producing high amounts of ROS, trig-gers multiscale alterations across biological samples that al-ter the homeostasis of oxidative processes
To ensure this point is clear to readers, consider saying, "The use of light irradiation is necessary to excite fluorescence but also produces ROS. In addition to inducing oxidative stress, ROS can also trigger multiscale alterations..."
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ncontrolled ROS processes
I might reword it to, "if oxidative processes are unchecked, they can lead to oxidative stress and cellular damage..."
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AI-enhanced intelligent microscopy
Is this the term that you want to introduce here or is it deep-learning augmented microscopy? It is a little hard for the reader to know which is the new term.
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The biological validity of live-cell imaging experiments re-quires a precise balance between the data quality and thespecimen’s health, as depicted in Fig. 1
This is an excellent point and a crucial argument for deep learning augmented microscopy.
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- Aug 2023
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we think a more likely explanation is that greater education and outreach, as well as continued improvements in installation and access, will be needed before end users trust these tools enough to become excited about applying them to their own work.
This is an interesting potential explanation for this decrease in interest in deep learning. Can you expand upon why you suspect this is the more likely explanation? Anything from the survey that supports this?
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‘written tutorials’ are highly preferred by the imaging community
I found this surprising at first and very helpful to know. It informs the level of detail that we should strive for when describing analysis methods.
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Figure 1
This figure is a bit pixelated making it hard to see the text and numbers. If the resolution can be improved, it will help the reader want to study the figure.
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- Jul 2023
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www.biorxiv.org www.biorxiv.org
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Multivariate details
This is useful to expand on the details of the statistical methods.
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The embedded images in the Multivariate details section did not load on my screen.
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we create Eq. scores for all 303 compounds in the dataset and use them as new features
What kind of time cost is associated with the training given the scale of this dataset?
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- Jun 2023
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www.biorxiv.org www.biorxiv.org
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We were able to collect spectral and spatial information of an identical tissue section in a robust and reliable manner, without compromising the quality of either.
This is a huge win for this technique to be able to get both Raman spectra and mass spec data from the same biological sample.
Do the peaks identified in the Raman-mapping inform any parameters for the subsequent MALDI imaging or is it that those peaks provide a basis for the segmentation.
If the biological sample has autofluorescence with 532 nm excitation and one must use a red-shifted laser for the Raman measurements, is there any reason why that would interfere with the MALDI analysis step?
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www.biorxiv.org www.biorxiv.org
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IAMSAM is a user-friendly web-based tool designed to analyze ST data
This seems like a very useful tool! I think it would be valuable for the reader if the authors comment on how the tool described in this study differs from or improves upon the 10X Visium software tools that accompany this type of ST dataset.
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www.biorxiv.org www.biorxiv.org
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Figure 1.
This figure shows up as a bit pixelated when enlarged. If it can be swapped with one at higher res, that would be useful.
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three primary functions of these segmentation notebooks
This is useful for breaking down the functions
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- May 2023
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www.biorxiv.org www.biorxiv.org
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This is an elegant study that experimentally untangles the role of a plant hormone in fungal colonization and development in host plants. The model proposed based on this work is very thorough as well.
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- Apr 2023
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www.biorxiv.org www.biorxiv.org
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Beautiful work! It is likely that this will be resolved with the journal submission, but just to watch out for the resolution on Fig 2-6, which is too low for viewing in this format.
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We
No italics needed
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)
extra ) here
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www.biorxiv.org www.biorxiv.org
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enlargement
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A very interesting computer-learning technique to improve signal to noise and enhance resolution of sub-cellular structures. The impact of this work will be more directly transmitted with clearer writing. Some specific suggestions are annotated throughout the paper.
One tool that might facilitate this is: https://hemingwayapp.com/
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bonds
should this be "bounds"?
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efficacious
perhaps "effective" is more direct
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requiring no clean data as references and no compromise of spatiotemporal resolution on diverse imaging systems
without the need for baseline data or loss of spatiotemporal resolution
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The computationally 15-fold multiplied photon budget in a standard confocal microscope by DeepSeMi allows for recording organelle interactions in four colors and high-frame-rate across tens of thousands of frames, monitoring migrasomes and retractosomes over a half day, and imaging ultra-phototoxicity-sensitive Dictyostelium cells over thousands of frames, all faithfully and sample-friendly.
consider making this a simpler, declarative statement(s)
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confocal microscope,
may want to specify the type of confocal here in addition to the methods.
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higher NA objective suffers even more from scattering
can you clarify how an objective with increased resolution (higher NA) leads to more scattering?
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generality
generalizability might be better here
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all faithfully
awkward phrasing
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to be
should be "from being"
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enlarged
increased is a better word choice than enlarged. Enlarged means to make bigger in size not magnitude. Increased is more accurate when talking about a larger number.
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clean data
more precise to say "data with high SNR"?
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and bio-friendly breaking the shot-noise limit, facilitating automated analysis of massive data about cell migrations and organelle interactions.
simplify and make more direct
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brand-
can eliminate to reduce redundancy
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fluctuated
what does fluctuated mean here?
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Besides,
Eliminate this.
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the contamination inevitable
specify the type of contamination
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roots in
odd phrasing here. What is more direct verb that can be used here?
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Fig. 4
Excellent figure to demonstrate the improvement in resolution with DeepSeMi over the raw images.
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no-compromise results
can no-compromise results be defined clearly?
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commercial
what kind?
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ten folds
tenfold
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DeepSeMi
Use of DeepSeMi
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colors
suggest saying "fluorophores in different channels. They were: tagBFP-SKL (Ex/Em 402/457 nm),..." to give the excitation and emission spectra for each fluorophore.
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in nonlinear microscope
using nonlinear microscopy or with a nonlinear microscope (and then specify which type)
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dye concentration
which dye concentration was better?
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enlarging excitation dosage
"increasing laser power" ?
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brittle
what does brittle mean here?
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magnificence of the harmonically orchestrated systems, organs, tissues, and cells
awkward phrasing. Perhaps something like "magnificently well-orchestrated systems of organs, tissues, and cells" or
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vehemently enlarged
"drastically improved" would be better
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Compared to recently developed interpolation-based denoising methods, DeepSeMi is capable of observing organelles of sophisticated movements and transformations without motion artifacts
Great point, may be stronger if framed as, "DeepSeMi improves upon interpolation-based denoising methods by (fill in the blank to describe what it is based on), which allows for observation of organelle dynamics without motion artifacts"
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high-frame-rate
revise use of hypens throughout. three hyphens should not be used consecutively.
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lustrous and remarkable advances in enriched fluorescence microscope[1, 9–12] have brought flourishing discoveries
awkward phrasing. Not sure what "enriched fluorescence microscope" really means
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100 ×
100x
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36,000 shots in multicolor
not a standard way to report this.
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Excitation power was set at 2% t
for which laser?
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Such gorgeous patterns reflect live organelles of complex, dynamic, and interplay in highly dynamic yet organized interactions capable of orchestrating complex cellular functions
awkward phrasing. Perhaps it would be better to clearly make the point that organelle interactions are dynamic and studying them with high spatiotemporal resolution is important to understand their function in the cell.
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but either damaging the fragile living systems or poisoning the cellular health and both altering morphological and functional interpretations that follow.
perhaps "but this practice can increase phototoxicity to the live specimens and disrupt the normal morphology" would be more clear
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Thirdly, data-hungry nature ensued
awkard phrasing
-
enables tens of times photon budget increments
computationally enables a tenfold increase in the photon budget
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holding a safe head
awkward phrasing
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manifolds
"multifold"
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differentiated ergo between
Not sure that "ergo" adds much meaning here. If it is wanted here then the punctuation should be, "differentiated; ergo, between"
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videography
Is it really videography? Is there a more precise term that can be used here?
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much finer temporal scal
any quantitative estimate on the improvement from previous methods?
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50 times fluorescent dye concentratio
the concentration of fluorescent dyes 50-fold
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0.53 μm/s and 0.46 μm/s,
assuming these are average rates, is there a +/- standard deviation that can be reported?
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14.6 μW (0.5% of 488 nm)
Very impressive with such gentle excitation
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Rather scattered tissues of C. elegans exuberate the noise contaminations
Can this be clarified?
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We applied DeepSeMi to circumvent the problem, which enabled dual-color and high-SNR imaging at the 45.3 μW dosage at 488 nm and the 49.8 μW dosage at 561 nm over 30 minutes without apparent photodamage
Great use of specific language here.
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helps better observation of structures and functions of cells and organisms in vivo
is this implying that because of their transparency, there is decreased background/noise? Perhaps this should be clarified.
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www.biorxiv.org www.biorxiv.org
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observed chromatin features that could shed light on how transcriptional activity takes place in organisms where chromosomes are permanently condensed
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numerous (up to 200) chromosomes
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cryo-immobilized by high-pressure freezing within 2 hours after collection
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these organisms can have photosensitive structures, called eyespots
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stay permanently condensed throughout their cell cycle
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small molecules live dyes
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putative photosensitive structure
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flagella, characteristic structures of dinoflagellates and at the associated eyespo
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globular shape in the center of the cell and more defined patches on the cell periphery
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confirm our prediction and identify the species by the thecal organization as Ensiculifera tyrrhenica
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2 flagella, protruding from basal bodies
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exogenously expressed fluorescent proteins
-
ogether with the 488nm excitation channel, an image of the transmitted laser light was generated using the T-PMT detector of the microscop
What was the autofluorescence excited by 488 nm indicating in the samples?
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and identify damaged cells to exclude from downstream processing
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We observed that the far-red signal matched with the position of the chloroplast and the nucleus
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implementing culture independent methods to study planktonic cells in their native ecosystem is truly important to better understand the cellular biology of these ecologically relevant microorganisms
This is an excellent point and justification for this study.
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Then we removed the measured thickness of resin present above the cell of interest with a trimming knife mounted on an ultramicrotome in three iterations of imaging and trimming.
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and identify damaged cells to exclude from downstream processing
What indicates cell damage from 488 nm autofluorescence?
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branded landmarks on the block surface using a near infrared (NIR, “2-photon”) laser in order to later facilitate its targeting at the FIB-SEM
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correlating 3D light and EM data beyond targeting purposes. The subcellular precision of such correlation allowed us to assign the emission of a fluorescent signal (far-red) to a specific organelle (chloroplast).
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While we cannot explain the autofluorescence in the nucleus, the overlap with the plastid is justified by the presence of chlorophyll
Interesting finding. Can any additional speculation be presented here?
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iji plugin MoBIE
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www.biorxiv.org www.biorxiv.org
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In
"An inVia Raman confocal microscope..."
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a locational information
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selecting the central points of the cells.
Were any measurements made across the cell using parameters for the maximal spatial resolution (0.25 µm)? It would be good to note (in a supplemental figure perhaps) that there was no difference across the 3 µm cell and justify using the center point of each cell for all of the measurements.
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(A) Schematic
Images would be useful prior to showing the measurements. Also, a title for the figure would be helpful.
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Statistical difference
What statistical test was used? It was not stated in the methods.
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a
remove a
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- Mar 2023
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www.biorxiv.org www.biorxiv.org
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a
remove a
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Statistical difference
What statistical test was used? It was not stated in the methods.
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(A) Schematic
Images would be useful prior to showing the measurements. Also, a title for the figure would be helpful.
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selecting the central points of the cells.
Were any measurements made across the cell using parameters for the maximal spatial resolution (0.25 µm)? It would be good to note (in a supplemental figure perhaps) that there was no difference across the 3 µm cell and justify using the center point of each cell for all of the measurements.
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In
"An inVia Raman confocal microscope..."
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- Feb 2023
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www.biorxiv.org www.biorxiv.org
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While we cannot explain the autofluorescence in the nucleus, the overlap with the plastid is justified by the presence of chlorophyll
Interesting finding. Can any additional speculation be presented here?
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and identify damaged cells to exclude from downstream processing
What indicates cell damage from 488 nm autofluorescence?
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implementing culture independent methods to study planktonic cells in their native ecosystem is truly important to better understand the cellular biology of these ecologically relevant microorganisms
This is an excellent point and justification for this study.
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ogether with the 488nm excitation channel, an image of the transmitted laser light was generated using the T-PMT detector of the microscop
What was the autofluorescence excited by 488 nm indicating in the samples?
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- Dec 2022
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www.biorxiv.org www.biorxiv.org
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50 times fluorescent dye concentratio
the concentration of fluorescent dyes 50-fold
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helps better observation of structures and functions of cells and organisms in vivo
is this implying that because of their transparency, there is decreased background/noise? Perhaps this should be clarified.
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Rather scattered tissues of C. elegans exuberate the noise contaminations
Can this be clarified?
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We applied DeepSeMi to circumvent the problem, which enabled dual-color and high-SNR imaging at the 45.3 μW dosage at 488 nm and the 49.8 μW dosage at 561 nm over 30 minutes without apparent photodamage
Great use of specific language here.
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holding a safe head
awkward phrasing
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14.6 μW (0.5% of 488 nm)
Very impressive with such gentle excitation
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much finer temporal scal
any quantitative estimate on the improvement from previous methods?
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0.53 μm/s and 0.46 μm/s,
assuming these are average rates, is there a +/- standard deviation that can be reported?
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Excitation power was set at 2% t
for which laser?
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enables tens of times photon budget increments
computationally enables a tenfold increase in the photon budget
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differentiated ergo between
Not sure that "ergo" adds much meaning here. If it is wanted here then the punctuation should be, "differentiated; ergo, between"
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videography
Is it really videography? Is there a more precise term that can be used here?
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36,000 shots in multicolor
not a standard way to report this.
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Compared to recently developed interpolation-based denoising methods, DeepSeMi is capable of observing organelles of sophisticated movements and transformations without motion artifacts
Great point, may be stronger if framed as, "DeepSeMi improves upon interpolation-based denoising methods by (fill in the blank to describe what it is based on), which allows for observation of organelle dynamics without motion artifacts"
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Thirdly, data-hungry nature ensued
awkard phrasing
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manifolds
"multifold"
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but either damaging the fragile living systems or poisoning the cellular health and both altering morphological and functional interpretations that follow.
perhaps "but this practice can increase phototoxicity to the live specimens and disrupt the normal morphology" would be more clear
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enlarging excitation dosage
"increasing laser power" ?
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lustrous and remarkable advances in enriched fluorescence microscope[1, 9–12] have brought flourishing discoveries
awkward phrasing. Not sure what "enriched fluorescence microscope" really means
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Such gorgeous patterns reflect live organelles of complex, dynamic, and interplay in highly dynamic yet organized interactions capable of orchestrating complex cellular functions
awkward phrasing. Perhaps it would be better to clearly make the point that organelle interactions are dynamic and studying them with high spatiotemporal resolution is important to understand their function in the cell.
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magnificence of the harmonically orchestrated systems, organs, tissues, and cells
awkward phrasing. Perhaps something like "magnificently well-orchestrated systems of organs, tissues, and cells" or
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100 ×
100x
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high-frame-rate
revise use of hypens throughout. three hyphens should not be used consecutively.
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DeepSeMi
Use of DeepSeMi
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in nonlinear microscope
using nonlinear microscopy or with a nonlinear microscope (and then specify which type)
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Fig. 4
Excellent figure to demonstrate the improvement in resolution with DeepSeMi over the raw images.
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vehemently enlarged
"drastically improved" would be better
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brittle
what does brittle mean here?
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fluctuated
what does fluctuated mean here?
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commercial
what kind?
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colors
suggest saying "fluorophores in different channels. They were: tagBFP-SKL (Ex/Em 402/457 nm),..." to give the excitation and emission spectra for each fluorophore.
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dye concentration
which dye concentration was better?
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no-compromise results
can no-compromise results be defined clearly?
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ten folds
tenfold
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enlarged
increased is a better word choice than enlarged. Enlarged means to make bigger in size not magnitude. Increased is more accurate when talking about a larger number.
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Besides,
Eliminate this.
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roots in
odd phrasing here. What is more direct verb that can be used here?
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generality
generalizability might be better here
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