Targeted volume correlative light and electron microscopy of an environmental marine microorganism
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Abstract
Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.
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Reply to the reviewers
Reviewer 1:
I am not an expert in algal ultrastructure or TEM, and I principally found this manuscript informative and well-written. I can only make minor recommendations, although I urge the Editors to supplement this review with others from specialists within the field.
We thank the reviewer for their positive comment on the manuscript as well as for the interesting following discussion points.
To date, to my knowledge, the Pentapharsodinium chloroplast has not been characterised at a molecular level. The placement of the nuclear lineage within the Peridiniales as a close relative or Durinskia spp. and other dinoflagellates with diatom …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer 1:
I am not an expert in algal ultrastructure or TEM, and I principally found this manuscript informative and well-written. I can only make minor recommendations, although I urge the Editors to supplement this review with others from specialists within the field.
We thank the reviewer for their positive comment on the manuscript as well as for the interesting following discussion points.
To date, to my knowledge, the Pentapharsodinium chloroplast has not been characterised at a molecular level. The placement of the nuclear lineage within the Peridiniales as a close relative or Durinskia spp. and other dinoflagellates with diatom endosymbionts raises the question of whether Pentapharsodinium possesses a peridinin-containing chloroplast, per most other dinoflagellates, or possesses a chloroplast of an alternative endosymbiotic derivation, although I suppose the single chloroplast would be typical for peridinin-containing chloroplasts from the Peridiniales. __Can the authors make any inference on this from their data? __
In the course of this study, we did not perform molecular characterization of the chloroplast. Therefore, formal answer to this question would not be possible based on our data.
However, we have evidence that suggest that the chloroplast from P. tyrrhenica does not come here from a diatom endosymbiont.
1/ In the suggested cases of diatom endosymbiont origin of the chloroplast, eg in the genus Durinskia, Yamada et al., 2019 show that Durinskia capensis or Durinskia kwazulunatalensis can present diatom organelles such as chloroplast, mitochondria, nucleus, or their remnants for a certain amount of time after uptake. The detailed analysis of our vEM data does not show these diatom organelles, thus ruling out the hypothesis.
2/ Additionally, the eyespot of dinoflagellate species possessing diatom association seems to be characteristic to these cells (Horiguchi., 2007, Hoppenrath et al., 2017). In our case, the eyespot of P. tyrrhenica corresponds to a distinct type (type IA, thought to be in peridinin dino-chloroplast according to Hoppenrath., 2017), that varies from the one observed in Durinksia baltica, or other dinoflagellates species possessing these diatom associations (Horiguchi., 2007, Hoppenrath et al., 2017).
Therefore, the chloroplast of our cell does not seem to be acquired from a diatom. However, as we don’t have molecular evidence at this stage, and are not able to perform such analyses, we prefer not to address this question in the manuscript.
While I appreciate that this is a study of a single cell only, I would prefer some more extensive evidence that the partial chromosome unfolding identified correlates to transcriptional activity. The nucleolus is surrounded by a layer of heterochromatin and perhaps the filamentous structures involved are transcriptionally quiescent. Were the authors able to take any preliminary images of cells harvested mid-day or exposed to higher light intensities, and do they see greater chromatin unfolding in this case? Similarly I would be curious if cells visualised later in the day possess multiple rather than single chloroplasts.
Our description is here based on one microorganism of very low abundance, and we do not have data for it across conditions. Therefore, we would not be comfortable inferring too many details in this paper and will thus modify the text in order be more careful about the potential role of these fibres and their putative association to transcription. We are planning to address this interesting point in the future, in a more biologically focused paper in preparation. Nevertheless, similar structures have been described protruding in the nucleoplasm in other species (Soyer., 1981, Bhaud et al., 2000, Decelle et al., 2021) and have been suggested to be associated to RNA transcription (Sigee., 1983). We will make sure to cite and discuss more literature on this point.
Line 39: should be "dinoflagellate biology"
Line 131: "a far red signal"
Line 196: should be "number of chloroplasts"We thank the reviewer, the errors will be corrected in the revised version.
Line 200: by curiosity, how does the measured chloroplast volume compare to those computed in vEM studies of Symbiodinium (c.f., Uwizeye 2021)
The measured chloroplast volume in our cell differs to those computed in Symbiodinium in Uwizeye et al., 2021. Indeed, in Uwizeye et al., 2021, the chloroplast represents 30% of the cell volume. This is more than in our cell for which the chloroplast represents only 9.5% of the cell volume. However, these two cells are different species and come from different growth conditions (culture VS environment). These factors potentially contribute to morphometric variations as the environment possesses different types and amounts of nutrients and light resources.
Line 221: how does the number of observed chromosomes compare to estimated chromosome numbers in dinoflagellates from karyotyping or whole-genome sequencing?
To our knowledge, there is a wide variability in terms of number of chromosomes observed between dinoflagellate species. Bhaud et al (2000) reports the presence of between 4 and 200 chromosomes depending on the species. Unfortunately, we did not find information concerning the number of chromosomes for this species to compare with our analysis. However, we are planning in the revised version to discuss how our workflow is complementary to other methods such as genomics, transcriptomics and light microscopy towards better understanding of marine organism.
Line 240: does the eyespot show any proximity to the mitochondria, as per the hybrid chloroplast/ mitochondrial-derived eyespots found in Warnowiacean dinoflagellates?
In our study the eyespot is composed of pigment globules, organized as a sheet, located inside the chloroplast and facing towards the theca. This type of arrangement seems to be distinct to the chloroplast/mitochondria-derived eyespot described in Warnowiaceae (Colley and Nilson., 2016). Additionally, for the revision of this paper we will further segment the mitochondria to investigate any potential proximity to the eyespot. We will also put the eyespot of P. tyrrhenica in context with other types described in the literature.
Reviewer #1 (Significance (Required)):
The application of vEM to environmental algal samples has to my knowledge not been attempted previously. If these approaches could be scaled up to a multi-cell approach and is not completely destructive to the cells, it could provide a fascinating way to connect algal morphology in the wild to other culture-free methods to understand algal biology (e.g., meta-barcoding).
We would like to address the interesting comment concerning the possibility to combine vEM along with other molecular tools to study organisms from a culture free method. While FIB-SEM is a destructive method, it could be used in combination with other methods and thus synergize towards the characterization of the environmental samples. We will add a few sentences in the discussion, as a forward look, as it represents indeed an important axis of our future research, where molecular taxonomy (e.g. metabarcoding) will be correlated to morphological characterisations. We believe such approaches will be useful beyond the study of micro-algae, and will make this point clear in the discussion.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This is avery important and interesting contribution to the ultrastructural analysis of one scientific species among many others living together in a rich sample as a marine microplankton. In addition the work also shows the possibility to obtain fundamental volumetric informations on the various structures and organelles. The work was well planned and executed and certainly represents a tremendous effort of the members of the group. It is clearly written, explaining each detail of the methodology necessary to the understanding of the whole work.
Reviewer #2 (Significance (Required)):
This a phantatis piece of scientific work where the authros were able to use the moderns three dimensional reconstruction technique possible using high resolution scanning electron to reconstruct one specific cell in a large population of heterogenous cells. The identification of one specific cell based on fluorescence images detected in a light microscopy was very important to present one new methodology to observed such types of cells. In addition to a detailed description of the strucutres and organelles found they were able to determne the area/volume occupies by each of them in cells.
Therefore I strong recommend the acceptance of the manuscript as it is.We thank the reviewer for their appreciation of our work.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary --
The authors present a workflow to characterize a microorganism, dinoflagellate cell, from environmental samples by vEM. The workflow enables the identification of specific taxa of interest in a heterogenous environmental sample and allows correlative fluorescence and FIB-SEM acquisition. I see no major flaws with this study. The authors present a proof of principle pipeline for utilizing CLEM to explore the ultrastructure of microorganisms.
Major comments --
What is not clear, is how highly reproducible this workflow is. For example, what is the time required for each sample? How manual vs automated is each step? While, the workflow is important and sound, it would be helpful for the reader to understand a little more about the variability and throughput. It doesn't need to be exhaustive, but further characterization of the workflow would significantly improve the impact of the manuscript and should be included. As a reader, it would be tremendously helpful to clearly state what is part of the preparation/imaging workflow and what is an application example of the workflow. If it is intended that the post-processing is also part of the workflow, there should be significantly more details provided into the segmentation and analyses processes.
We thank the reviewers for pointing this out. This workflow has been applied to several blocks and repeated imaging of a subset of species could be achieved with 100% success. We will show this in a follow up paper. Moreover, the same method has been applied for targeted FIB-SEM acquisition of samples expressing fluorescent proteins (Ronchi et al., 2021). Therefore, we believe our workflow is highly reproducible and robust. We are planning to address the concern on technical variability in our revised version, together with a better explanation of time scales and automated VS manual aspect of the steps that are used for this workflow. We will also add information concerning the segmentation.
Minor comments --
As this study describes a workflow that was developed for identification and imaging of microorganisms, it would be highly beneficial to the reader to have a figure that shows each of the steps, end to end.
We thank the reviewer for this suggestion. We plan to add a supplementary figure that describes the workflow step by step.
It would also be helpful to add annotation labels and a scale bar to supplemental video 2.
We thank the reviewer for spotting this missing information. We plan to add annotation labels as well as a scale bar to the video S2.
Do the nonphotosynthetic species also have nuclear autofluorescence or is this just a trait of a subset of the photosynthetic species?
We thank the reviewer for this question. From our analysis, we can’t conclude that it is a trait of some photosynthetic species compared to non-photosynthetic ones. However, heterogeneous autofluorescence profiles, even though unexplained, represent a valuable tool to discriminate between cell types. We would like to further address this point in the manuscript that such fluorescence profiles, even though unexplained, can contribute to the identification of specific cell types.
Reviewer #3 (Significance (Required)):
General assessment --
The authors present an important, yet missing, workflow for characterizing microorganisms from environmental samples using vEM and CLEM. A strength of the study is that it enables identification and selection of taxa in heterogenous samples. Using correlative, confocal imaging of both auto-fluorescence and transmitted light, the authors show that specific taxa can be identified and selected for according to the organism's photosynthetic and morphological properties.
We thank the reviewer for their positive comments.
To improve upon this, it could be useful for the authors to provide a list or table of potential organisms that could be selected for in this manner to exemplify the use cases of this protocol.
We thank the reviewer for their suggestion. We plan to add a supplementary figure with a gallery of different cells and their identification.
Advance --
This study, while a proof of concept, is also one of the first examples of using vEM on environmental studies. The author's also present the potential value add of using CLEM, not just for selection purposes, but also more comprehensive identification and mapping of subcellular structures. While the workflow itself is incremental (and important), the application is quite novel.
Audience --
Because the authors' study combines a vEM workflow and microorganism characterization, the potential audience is broad reaching. The workflow itself could be adapted to many different systems - beyond microorganisms - making it of use to several biological fields.
We thank the reviewer for their positive comments.
Expertise --
vEM, CLEM, FIB-SEM, membrane trafficking, cell biology, tissue cell biology, machine learning
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #3
Evidence, reproducibility and clarity
Summary
The authors present a workflow to characterize a microorganism, dinoflagellate cell, from environmental samples by vEM. The workflow enables the identification of specific taxa of interest in a heterogenous environmental sample and allows correlative fluorescence and FIB-SEM acquisition. I see no major flaws with this study. The authors present a proof of principle pipeline for utilizing CLEM to explore the ultrastructure of microorganisms.
Major comments
What is not clear, is how highly reproducible this workflow is. For example, what is the time required for each sample? How manual vs automated is each step? While, the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary
The authors present a workflow to characterize a microorganism, dinoflagellate cell, from environmental samples by vEM. The workflow enables the identification of specific taxa of interest in a heterogenous environmental sample and allows correlative fluorescence and FIB-SEM acquisition. I see no major flaws with this study. The authors present a proof of principle pipeline for utilizing CLEM to explore the ultrastructure of microorganisms.
Major comments
What is not clear, is how highly reproducible this workflow is. For example, what is the time required for each sample? How manual vs automated is each step? While, the workflow is important and sound, it would be helpful for the reader to understand a little more about the variability and throughput. It doesn't need to be exhaustive, but further characterization of the workflow would significantly improve the impact of the manuscript and should be included.
As a reader, it would be tremendously helpful to clearly state what is part of the preparation/imaging workflow and what is an application example of the workflow. If it is intended that the post-processing is also part of the workflow, there should be significantly more details provided into the segmentation and analyses processes.
Minor comments
As this study describes a workflow that was developed for identification and imaging of microorganisms, it would be highly beneficial to the reader to have a figure that shows each of the steps, end to end.
It would also be helpful to add annotation labels and a scale bar to supplemental video 2.
Do the nonphotosynthetic species also have nuclear autofluorescence or is this just a trait of a subset of the photosynthetic species?
Significance
General assessment
The authors present an important, yet missing, workflow for characterizing microorganisms from environmental samples using vEM and CLEM. A strength of the study is that it enables identification and selection of taxa in heterogenous samples. Using correlative, confocal imaging of both auto-fluorescence and transmitted light, the authors show that specific taxa can be identified and selected for according to the organism's photosynthetic and morphological properties. To improve upon this, it could be useful for the authors to provide a list or table of potential organisms that could be selected for in this manner to exemplify the use cases of this protocol.
Advance
This study, while a proof of concept, is also one of the first examples of using vEM on environmental studies. The author's also present the potential value add of using CLEM, not just for selection purposes, but also more comprehensive identification and mapping of subcellular structures. While the workflow itself is incremental (and important), the application is quite novel.
Audience
Because the authors' study combines a vEM workflow and microorganism characterization, the potential audience is broad reaching. The workflow itself could be adapted to many different systems - beyond microorganisms - making it of use to several biological fields.
Expertise
vEM, CLEM, FIB-SEM, membrane trafficking, cell biology, tissue cell biology, machine learning
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This is avery important and interesting contribution to the ultrastructural analysis of one determoned species among many others living together in a rich sample as a marine microplankton. In addition the work also shows the possibility to obtain fundamental volumetric informations on the various structures and organelles. The work was well planned and executed and certainly represents a tremendous effort of the members of the group. It is clearly written, explaining each detail of the methodology necessary to the understanding of the whole work.
Significance
This a phantatis piece of scientiic work where the authros were able to …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This is avery important and interesting contribution to the ultrastructural analysis of one determoned species among many others living together in a rich sample as a marine microplankton. In addition the work also shows the possibility to obtain fundamental volumetric informations on the various structures and organelles. The work was well planned and executed and certainly represents a tremendous effort of the members of the group. It is clearly written, explaining each detail of the methodology necessary to the understanding of the whole work.
Significance
This a phantatis piece of scientiic work where the authros were able to use the moderns three dimensional reconstruction technique possible using high resolution scanning electron to reconstruct one specific cell in a large population of heterogenous cells. The identification of one specific cell based on fluorescence images detected in a light microscopy was very important to present one new methodology to observed such types of cells. In addition to a detailed description of the strucutres and organelles found they were able to determne the area/volume occupies by each of them in cells. Therefore I strong recommend the acceptance of the manuscript as it is.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
I am not an expert in algal ultrastructure or TEM, and I principally found this manuscript informative and well-written. I can only make minor recommendations, although I urge the Editors to supplement this review with others from specialists within the field.
To date, to my knowledge, the Pentapharsodinium chloroplast has not been characterised at a molecular level. The placement of the nuclear lineage within the Peridiniales as a close relative or Durinskia spp. and other dinoflagellates with diatom endosymbionts raises the question of whether Pentapharsodinium possesses a peridinin-containing chloroplast, per most other …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
I am not an expert in algal ultrastructure or TEM, and I principally found this manuscript informative and well-written. I can only make minor recommendations, although I urge the Editors to supplement this review with others from specialists within the field.
To date, to my knowledge, the Pentapharsodinium chloroplast has not been characterised at a molecular level. The placement of the nuclear lineage within the Peridiniales as a close relative or Durinskia spp. and other dinoflagellates with diatom endosymbionts raises the question of whether Pentapharsodinium possesses a peridinin-containing chloroplast, per most other dinoflagellates, or possesses a chloroplast of an alternative endosymbiotic derivation, although I suppose the single chloroplast would be typical for peridinin-containing chloroplasts from the Peridiniales. Can the authors make any inference on this from their data?
While I appreciate that this is a study of a single cell only, I would prefer some more extensive evidence that the partial chromosome unfolding identified correlates to transcriptional activity. The nucleolus is surrounded by a layer of heterochromatin and perhaps the filamentous structures involved are transcriptionally quiescent. Were the authors able to take any preliminary images of cells harvested mid-day or exposed to higher light intensities, and do they see greater chromatin unfolding in this case? Similarly I would be curious if cells visualised later in the day possess multiple rather than single chloroplasts.
Finally, I have a few (very) small grammatical corrections:
Line 39: should be "dinoflagellate biology"
Line 131: "a far red signal"
Line 196: should be "number of chloroplasts"
Line 200: by curiosity, how does the measured chloroplast volume compare to those computed in vEM studies of Symbiodinium (c.f., Uwizeye 2021)
Line 221: how does the number of observed chromosomes compare to estimated chromosome numbers in dinoflagellates from karyotyping or whole-genome sequencing?
Line 240: does the eyespot show any proximity to the mitochondria, as per the hybrid chloroplast/ mitochondrial-derived eyespots found in Warnowiacean dinoflagellates?
Significance
The application of vEM to environmental algal samples has to my knowledge not been attempted previously. If these approaches could be scaled up to a multi-cell approach and is not completely destructive to the cells, it could provide a fascinating way to connect algal morphology in the wild to other culture-free methods to understand algal biology (e.g., meta-barcoding).
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peer
I think you mean "pier".
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UA
Uranyl acetate acronym is undefined.
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The correlative light and electron microscopy data in this work is absolutely amazing! I'm completely blown away by the isotropic and featureful volumetric segmentation! I wish I had more substantive and constructive feedback to provide, but I'm just impressed with the high the quality of the imaging data. I very much enjoyed looking through the data in the manuscript.
I have one major comment. I couldn't find the data on EMPIAR. I guess it hasn't been released publicly yet?
Besides that one comment I just have some minor, mainly editorial and stylistic, suggestions.
Watch out for acronyms. Make sure to define them so the reader can quickly understand what they are reading or looking at. For example, 2p-branding is not defined in the Figure 2 legend. I suggest sticking to either the 2p-branding or the NIR branding designation. They are …
The correlative light and electron microscopy data in this work is absolutely amazing! I'm completely blown away by the isotropic and featureful volumetric segmentation! I wish I had more substantive and constructive feedback to provide, but I'm just impressed with the high the quality of the imaging data. I very much enjoyed looking through the data in the manuscript.
I have one major comment. I couldn't find the data on EMPIAR. I guess it hasn't been released publicly yet?
Besides that one comment I just have some minor, mainly editorial and stylistic, suggestions.
Watch out for acronyms. Make sure to define them so the reader can quickly understand what they are reading or looking at. For example, 2p-branding is not defined in the Figure 2 legend. I suggest sticking to either the 2p-branding or the NIR branding designation. They are used interchangeably in the text.
I suggest placing the scale bar in images in the first panel, instead of the last panel. See Figure 3. The reader will look at the figure from left to right, not right to left.
It would be helpful to the reader to label the different features that are shown in supplementary video 2.
Could you show a 3D rendering of all of the features shown in supplementary video 2? Currently individual components are rendered separately, but it would be useful to the reader to see the relative positions of the cellular components.
Could you speculate on what cellular components are being visualized by the green autofluorescence?
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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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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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and identify damaged cells to exclude from downstream processing
What indicates cell damage from 488 nm autofluorescence?
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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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UA
Uranyl acetate acronym is undefined.
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peer
I think you mean "pier".
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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?
-
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?
-
The correlative light and electron microscopy data in this work is absolutely amazing! I'm completely blown away by the isotropic and featureful volumetric segmentation! I wish I had more substantive and constructive feedback to provide, but I'm just impressed with the high the quality of the imaging data. I very much enjoyed looking through the data in the manuscript.
I have one major comment. I couldn't find the data on EMPIAR. I guess it hasn't been released publicly yet?
Besides that one comment I just have some minor, mainly editorial and stylistic, suggestions.
Watch out for acronyms. Make sure to define them so the reader can quickly understand what they are reading or looking at. For example, 2p-branding is not defined in the Figure 2 legend. I suggest sticking to either the 2p-branding or the NIR branding designation. They are …
The correlative light and electron microscopy data in this work is absolutely amazing! I'm completely blown away by the isotropic and featureful volumetric segmentation! I wish I had more substantive and constructive feedback to provide, but I'm just impressed with the high the quality of the imaging data. I very much enjoyed looking through the data in the manuscript.
I have one major comment. I couldn't find the data on EMPIAR. I guess it hasn't been released publicly yet?
Besides that one comment I just have some minor, mainly editorial and stylistic, suggestions.
Watch out for acronyms. Make sure to define them so the reader can quickly understand what they are reading or looking at. For example, 2p-branding is not defined in the Figure 2 legend. I suggest sticking to either the 2p-branding or the NIR branding designation. They are used interchangeably in the text.
I suggest placing the scale bar in images in the first panel, instead of the last panel. See Figure 3. The reader will look at the figure from left to right, not right to left.
It would be helpful to the reader to label the different features that are shown in supplementary video 2.
Could you show a 3D rendering of all of the features shown in supplementary video 2? Currently individual components are rendered separately, but it would be useful to the reader to see the relative positions of the cellular components.
Could you speculate on what cellular components are being visualized by the green autofluorescence?
-
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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