In these examples, the lifetime of pixels representing autofluorescence and GFP are quite different, which implies clearly distinct clusters in the phasor plots.
This is quite helpful. I have observed this autofluorescence especially excited around 483 nm and now will be able to separate it by lifetime.
showing two the two fluorescence
Fig. 4 caption. I believe this is a typo "two the two" that is repeated in (V) also.
The community-driven development of next-generation bioimage file formats (https://ome-model.readthedocs.io/en/stable/developers/6d-7d-and-8d-storage.html) will likely simplify the handling of FLIM datasets along the data life cycle including visualization, integrative lifetime analysis and sharing according to the FAIR principles in the future.
This is a great step towards better integration of microscopy images with analyzed data in a way that retains the spatial context for the viewer.
Very interesting work to reveal immunomodulatory mechanisms of Lactobacillus species in the vaginal microbiome.
The background on the loss of espBCD genes in many Lactobacillus species and retention of espA in host-associated taxa is very interesting. It would be good to reference Figure 1 after lines 116-118.
In the future, it would help the reader to label each figure as they appear within the text to make it easier when cross referencing the figure legends at the end.
In Figure 2, the TEM micrographs comparing the seem show that WT have more EPS, but the dark cloud around the EPS mutant makes it hard to tell if it's more EPS or none. The methods say that three micrographs were analyzed, but panel B shows n=2. It would be good to clarify the number of cells per image and if it was quantified by area of the dark cloud around the cell.
The use of confocal microscopy and SEM really shows how the bacteria interact with the monolayer of cells (Figure 5). Did you consider using spent media from WT and EPS mutant cells as a negative control. What was measured from these images? The cell layer in c and f certainly appear different, but the bacteria seem to be more abundant than in b and e with the WT strain.
Moreover, the fluorophore attached to palmitic acid significantly increases cytotoxicity than dPA and photobleaching over time.
How was cytotoxicity measured and compared to cells with dPA to ensure the Raman probes were not also cytotoxic?
wide spread of concentration value s
there is a typo here, it should say "values".
The first step was to prepare a calibration curve for RS. Deuterated fatty acid was dissolved in various concentrations of oleic acid and, after being applied to a CaF2 slide, spectra were measured for selected concentrations ranging from 0 mM to 3477 mM for dPA.
These two sentences are repeated in this section.
However, the lack of a large diel venting cycle in the S. affinis light organ may be the reason for the absence of the bottleneck, without a need to suddenly release the majority of the bacterial population.
The fact that there are twelve crypts co-colonized by strains with different temperature sensitivities is fascinating. I wonder how the crypts prevent non-specific strains from colonizing them. Perhaps the biochemistry of the migration pathway is especially harsh and/or phagocytotic immune cells are patrolling for non-Vibrio bacteria.
Riboflavin plays an important role in women’s health due to increased physiological demands linked to menstruation, fertility, pregnancy, and pelvic tissue integrity and is implicated in neurological and psychiatric conditions such as postpartum depression, and migraine
Do Lactobacillus species become reduced in the vaginal microbiome during pregnancy? It would be interesting to follow up on this work to understand how the host-microbe exchange of metabolites changes in pregnancy-shifted microbiomes.
Our observation that exposure to riboflavin-overproducing lactobacilli significantly enhances 5,6-diMe-Ben, all trans retinal, coenzyme Q10, PABA, β-NAD and reduced glutathione in vaginal epithelial cells suggests a direct contribution of Lactobacillaceae to vaginal host redox homeostasis.
Do you suspect that these compounds originate exclusively from the lactobacilli in the vagina, or is it possible that some contribution is from microbial activity in the gut? Because there was no correlation between vitamin intake and the presence of vaginal lactobacilli, perhaps it's unlikely that the gut microbiome is contributing.
7, ?, 47],
There is a question mark instead of a linked citation here.
For instance, increased snacking and carbohydrate intake during the first trimester (T1) may favor cariogenic bacteria, while micronutrient supplementation (e.g., folic acid, iron) may mitigate dysbiosis
Did the study track whether the women were taking a prenatal vitamin with sufficient folic acid? I wonder if this mitigated any of the dysbiosis observed across the three trimesters. Is it known that the microbial taxa that were lost are particularly sensitive to iron and folate availability?
group
small typo: "groups"
evidence age-specific
small typo: "evidence of"
A recent study [14] compared bacterial communities in formula- and breast-fed infants, clock-time based assessments revealed this rhythm, yet without accounting for the timing of stool samples in relation to other potential zeitgebers, which may not fully capture drivers of microbial rhythmicity.
How did the present study account for the type of food (formula-, breast-, and combo-fed) in infants or the timing of introduction of solid food?
For repeated measures,
it is a little unclear whether some individuals were sampled at multiple ages. The description in the methods describes them as independent cohorts, but repeated measures are mentioned here.
Specifically, we highlight a microbial diversity increase with longer wake periods, indicating that microbial diversity benefits from resumed physiological processes during the waking period
What type of physiological processes might increase microbial diversity during infant wakefulness? Do you think that longer wakefulness is correlated with higher sampling of the environment (ingesting microbes) and that increases microbial diversity?
This study
It was interesting that two samples from CMB clustered with A. exaltata in the PCA shown in Fig. S6. What do you make of that, given that those samples came from a site isolated from the others?
A small note is that the figure caption for Fig. S4 appears incorrect and is the same for Fig. S6
Abstract
I found it fascinating that the two populations of milkweed have such different reproductive microbiomes and that the bacteria isolated from A. syriaca may be negatively affecting pollination in the host. It would be interesting to follow up on the fungal and bacterial isolates to determine if 1) the yeast in each host differ dramatically and 2) if the yeast from A. exaltata are outcompeting bacteria or directly inhibiting their survival in the host.
While we were primarily interested in the effect of the host on symbiont fitness, supplementing the free-living symbiont is also likely to benefit the host. In the first experiment, the final difference of ∼4,300 cells per gram represented a ∼75% increase compared to soils unexposed to ins
How a host affects symbiont fitness is an interesting area. Do you think that the dead hosts provide a food source specifically to the symbionts as they transition to the soil environment or to many soil microbes? Is there any reason to believe that the bacterial symbiont can utilize the dead bugs to outcompete other soil microbes?
2.1 Training Modes and Required Training Data
MicroSplit is an exciting technique that could drastically improve the feasibility of multiplexed imaging. I'm wondering if the amount of training data differs depending on the training mode. For example, does training mode III represent more of a ground truth because it's based on images of each channel separately and thus requires less data? Is that the preferred training mode when possible, or does the model perform equally well with all training modes?
Figure 1
The interpretability of this figure could be enhanced with some minor tweaks. 1) Labels for each column to distinguish plots from experiments 1, 2, or combined would be helpful if it is necessary to show all three plots for each variable. Alternatively, you could show only the combined experiments and put the plots for each experiment in the supplemental.
2) The plots show larval settlement in response to a given variable in two states: "sterilized" and "untreated." I do not see where in the methods or results these two conditions are described, and I found this confusing.
3) In addition, it's difficult to quickly get a sense of which factors significantly impacted larval settlement. Labeling the points with different letters invokes that they indicate significant differences determined by the Tukey's post hoc test. However, the table shows that many of these factors did not impact settlement as determined by the model. Making these distinctions very clear to the reader in a visual way would greatly enhance the impact of the results.
Predator cues did not decrease settlement significantly, however there was a non-significant trend showing a reduction of settlement
Is there any way to determine that the concentration of the predator cue was high enough to impact the oyster larvae? Is there another readout that could be used as a positive control to know that the larvae could sense the cue but did not respond?
This beautiful study highlights the power of SRS microscopy for discovering and following up on important observations. If addressed, I have a couple of questions that would provide clarity to the reader.
The inset graph showing the normalized pure spectra of proteins and lipids is critical for understanding the protein and lipid channels used throughout. I see where the normalization is explained in the methods, but I didn't find the details about how those curves were obtained. What was used for the pure protein and lipid samples? How many wavenumbers were sampled (just the 5 with the tick marks on the x-axis)?
In Figure 1, it would be helpful to add markings to point out the additional structures described in the figure caption for non-experts in C. elegans.
In Figure 2, the inset really demonstrates the protein buildup in the pseudocoelomic space. Did you notice that that region on the worm was particularly striking across treatments? Would comparing the quantification in those FOV show a more pronounced effect than the entire worm?
The schematic showing the experimental design in Fig 1G. is helpful, but I didn't see the reasoning in the text to explain why the daf-2 mutants weren't exposed to the calorie restriction treatment like for ceh-60 worms.
In Figure S1, were the protein or lipid quantifications ever normalized to the area of the worm?
What do you make of the decreased protein buildup in ceh-60 CR worms as compared to ceh-60 fed (Fig. 6F). I see that there was no additive effect of CR on the mutants in terms of lifespan, but I found it interesting that it did look significantly lower in this plot.
When virtual staining of the plasma membrane was applied to these images, live cells displayed clear and intact cell contours, while dead cells exhibited shrunken and collapsed internal cell membranes (Fig. 7a)
This is a cool application of virtual staining that would eliminate the need for a live-dead stain. Did you confirm the brightfield morphologies with a viability stain?
Circularity was defined as 4πSL–2, where S and L represent the cell area and perimeter, respectively. The highest value of 1 indicates a perfect circle, whereas the lowest value of 0 indicates a highly complex shape
Examining the cell's eccentricity may be interesting if it captures something not reflected in the circularity calculation.
561
Was 561 nm the optimal excitation for chloroplasts in this sample? I would expect the optimal excitation to be either between 400-460 nm or 600-750 nm.
UPlanXApo ×20; Olympus
A more standard way to report the objective would be "Olympus UPlanApo 20x"
These training and test images are publicly available
Were all images acquired on the same day, or were these compiled across multiple imaging days? Given the day-to-day variability that imaging experiments can have (sample prep and microscope performance), it would be worth noting the number of images acquired per day that ended up in the dataset. How was the splitting of images into training and test datasets determined?
These mixed tubules may result from direct interaction between community members, e.g. Alphaproteobacteria invading tubules of other bacteria, perhaps by migrating through the interstitial region between tubules or from the intergland space. This spatial localization has physiological implications: the high density at which Alphaproteobacteria bacteria inhabit tubules, along with their ability to cohabit with or invade tubules of different bacteria, likely accounts for the group’s predominance in the adult ANG bacterial community.
Is it possible to disturb the ANG microbial community by using antibiotics? If so, it would be interesting to look at how th populations rebound during the recovery phase.
Although tubules containing mixed populations were infrequent, they were observed in every biological replicate (n=5).
Do you suspect that populations within more tubules are mixed if you were able to resolve lower levels of phylogenetic classifications?
A peristaltic motion appears to push the tubule’s contents into the intergland space (Supplemental Video 4), and the cilia lining the NG and ANG in the intergland space may facilitate the mixing of bacteria and jelly as well as the transport to the egg.
This observation is intriguing. Do you think that this is a rare occurrence, given how only 50-75% of the ANGs were occupied by symbionts? Any ideas what might trigger the peristaltic motion and whether it could be triggered by environmental light, like the light organ venting process?
Figure 7
This model sheds new light on the biophysical processes involved in ANG microbiogeography. I noticed that protrusions are lacking from the epithelial cells lining the tubules deep in the ANG (Fig 7B). Do you have any insight into whether these epithelia are microvillous like the light organ crypts or ciliated like the preceeding portions of the ANG and intergland space?
These slides only had FISH probes targeting Alphaproteobacteria, Gammaproteobacteria, and Verrucomicrobia, hybridized as described above.
Were these slides sequential slices from the same ANG or were they ANGs from the five different animals?
COMBO necessarily brings about a different physiological state than TAP, and this difference may influence C. reinhardtii’s latent tendency to form clonal assemblages and/or extracellular matrices that cause bodies to settle rapidly in aqueous media.
It would be interesting to gain further insight into the physiological differences when grown in the two types of media.
We observed that multicellular cluster size varied among evolved strains over the course of 6-days.
Why do you think the multicellular cluster size decreased over time for 3 out of the 4 PS strains? Was this surprising or a sign of instability?
Next, images were manually screened to demarcate cluster boundaries and record cell number within each boundary using Cell Counter in ImageJ (https://imagej.nih.gov/ij/). Cell number was estimated by counting DAPI stained nuclei confined within cluster boundaries (e.g., Figure 2).
These cluster boundaries look hard to delineate by eye. I wonder if you considered using a stain (like Calcafluor white) to make the cell walls more obvious to the observer.
Multicellular structures evolved under predation-selection but not under settling selection
It would help orient the reader to have a visual representation of this part of the results. A schematic could show that multicellular phenotypes emerged at different times, but a subset of those that evolved under the predator-selective regime were stable. This could have the strain names with the stable multicellular ones highlighted so the reader can easily reference this figure later when looking at the results that follow up on those strains (like Figs. 3 & 4).
Microscopic images
It's more accurate to call these micrographs rather than microscopic images.
Results
The first few paragraphs of the results section are more suitable for the introduction. I would consider revising the introduction and integrating the information presented as results to better prepare the reader for this study's real results.
Fig. 6
It is hard to extract much information from Fig 6A with how the spectra are currently shown. It may be more useful to show each spectrum separately and indicate the corresponding organelle. Then, indicate the three sections of the spectrum that are shown in 6B-D.
Figure 2
Is the y-axis showing intensity normalized?
As illustrated in Table S1 in the supplementary information, 38 peaks common to all examined EPs were identified.
How did you identify these peaks? Was it based on visual observation of differences in intensity across wavenumbers, or was there a more analytical approach taken?
The cells were observed with a Nikon microscope equipped with a Nikon DS-Qi2 camera
What light source was used and what was the duration of the videos for the motility assay? Given their negative phototaxis, I'm wondering if red light was used for imaging.
resuspended in nitrogen-free HSM media to trigger gametogenesis and enhance flagellar movement.
How long were the cells incubated in the nitrogen-free HSM medium before the phototaxis and motility assays? We've observed variation in gametic motility depending on the time spent in water prior to the assay.
first 10 Principal Components (PCs)
How many PCs can explain 95% of the variance? And how many features went into the PCA?
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?
Immune Cell
I don't think it's necessary to capitalize immune cell while naming the type of cluster.
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?
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?
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.
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).
Fig. 4
I see now that the label for Darkened should really be "Dark-adapted", is that true?
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.
may be observed with the naked eye
Interesting, what is observed by eye? Is it a darkening of a green hue?
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.
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.
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.
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.
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?
EDS1
Just a note for readers outside of the Arabidopsis field that EDS1 hasn't been defined yet in the abstract.
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.
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.
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.
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).
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?
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.
during the resolution phase
It would be helpful to the reader to hear more about how the resolution phase was determined.
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.
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.
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.
Pipeline II was deemed the most robust,
It would be helpful to hear more about how the level of robustness was determined.
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.
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.
mitohconria
typo, mitochondria
images
typo, should be imaged
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?
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.
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?
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?
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?
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.
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.
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.
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.
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.
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.
Given this, alongside time-consuming data an-notation processes, has
Revise this to ensure a proper subject.
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.
CLEM
define CLEM in this section
confocal mi-croscop
see point about Fig. 2B
Yet
Eliminate and start with "An"
less commonly employed
"rarer"
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.
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.
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.
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.
environment’
typo, eliminate the '
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)."
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.
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..."
ncontrolled ROS processes
I might reword it to, "if oxidative processes are unchecked, they can lead to oxidative stress and cellular damage..."
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.
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.
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?
‘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.
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.
Multivariate details
This is useful to expand on the details of the statistical methods.
The embedded images in the Multivariate details section did not load on my screen.
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?
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?
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.
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.
three primary functions of these segmentation notebooks
This is useful for breaking down the functions
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.
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.
We
No italics needed
)
extra ) here
enlargement
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/
bonds
should this be "bounds"?
efficacious
perhaps "effective" is more direct
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
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)
confocal microscope,
may want to specify the type of confocal here in addition to the methods.
higher NA objective suffers even more from scattering
can you clarify how an objective with increased resolution (higher NA) leads to more scattering?
generality
generalizability might be better here
all faithfully
awkward phrasing
to be
should be "from being"
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.
clean data
more precise to say "data with high SNR"?
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
brand-
can eliminate to reduce redundancy
fluctuated
what does fluctuated mean here?
Besides,
Eliminate this.
the contamination inevitable
specify the type of contamination
roots in
odd phrasing here. What is more direct verb that can be used here?
Fig. 4
Excellent figure to demonstrate the improvement in resolution with DeepSeMi over the raw images.
no-compromise results
can no-compromise results be defined clearly?
commercial
what kind?
ten folds
tenfold
DeepSeMi
Use of DeepSeMi
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.
in nonlinear microscope
using nonlinear microscopy or with a nonlinear microscope (and then specify which type)
dye concentration
which dye concentration was better?
enlarging excitation dosage
"increasing laser power" ?
brittle
what does brittle mean here?
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
vehemently enlarged
"drastically improved" would be better
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"
high-frame-rate
revise use of hypens throughout. three hyphens should not be used consecutively.
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
100 ×
100x
36,000 shots in multicolor
not a standard way to report this.
Excitation power was set at 2% t
for which laser?
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.
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
Thirdly, data-hungry nature ensued
awkard phrasing
enables tens of times photon budget increments
computationally enables a tenfold increase in the photon budget
holding a safe head
awkward phrasing
manifolds
"multifold"
differentiated ergo between
Not sure that "ergo" adds much meaning here. If it is wanted here then the punctuation should be, "differentiated; ergo, between"
videography
Is it really videography? Is there a more precise term that can be used here?
much finer temporal scal
any quantitative estimate on the improvement from previous methods?
50 times fluorescent dye concentratio
the concentration of fluorescent dyes 50-fold
0.53 μm/s and 0.46 μm/s,
assuming these are average rates, is there a +/- standard deviation that can be reported?
14.6 μW (0.5% of 488 nm)
Very impressive with such gentle excitation
Rather scattered tissues of C. elegans exuberate the noise contaminations
Can this be clarified?
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.
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.
observed chromatin features that could shed light on how transcriptional activity takes place in organisms where chromosomes are permanently condensed
numerous (up to 200) chromosomes
cryo-immobilized by high-pressure freezing within 2 hours after collection
these organisms can have photosensitive structures, called eyespots
stay permanently condensed throughout their cell cycle
small molecules live dyes
putative photosensitive structure
flagella, characteristic structures of dinoflagellates and at the associated eyespo
globular shape in the center of the cell and more defined patches on the cell periphery
confirm our prediction and identify the species by the thecal organization as Ensiculifera tyrrhenica
2 flagella, protruding from basal bodies
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?
and identify damaged cells to exclude from downstream processing
We observed that the far-red signal matched with the position of the chloroplast and the nucleus
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.
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.
and identify damaged cells to exclude from downstream processing
What indicates cell damage from 488 nm autofluorescence?
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
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).
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?
iji plugin MoBIE
In
"An inVia Raman confocal microscope..."
a locational information
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.
(A) Schematic
Images would be useful prior to showing the measurements. Also, a title for the figure would be helpful.
Statistical difference
What statistical test was used? It was not stated in the methods.
a
remove a
a
remove a
Statistical difference
What statistical test was used? It was not stated in the methods.