Usefulness of super-resolution?

[Csúcs Gábor-3]

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Dear All,

I would have a slightly provocative question to the community: Could you help me to identify significant/major biological discoveries that were clearly dependent on the availability of various super-resolution light microscopy methods? The PNAS paper from the Zhuang lab (2016) about the actin-spectrin ring is a good example for me but I am looking for further ones. Of course, we also use super-resolution techniques in our facility but my observation is that these are used rather to provide "one nice image for a publication" or "another piece in the puzzle of evidences" but they are not "game winners", they were not necessarily the major piece of evidence to prove a biological hypothesis. So I am looking for biological questions that could be answered "only"/mostly by the existing super-resolution methods.
        Thanks a lot for your help!

Greetings Gabor

[Jakub Chojnacki]

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Dear Gabor,

I realise that I am promoting my own work here, but here is a clear example
of a discovery of the novel mechanism important for HIV entry competence.
This work would not be possible without a super-resolution microscopy
approach (in this case STED).
http://science.sciencemag.org/content/338/6106/524

Hope this helps.

Regards,
Jakub

On 19 August 2017 at 16:46, Csúcs Gábor <[hidden email]> wrote:

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> *****
>
>
> Dear All,
>
> I would have a slightly provocative question to the community: Could you
> help me to identify significant/major biological discoveries that were
> clearly dependent on the availability of various super-resolution light
> microscopy methods? The PNAS paper from the Zhuang lab (2016) about the
> actin-spectrin ring is a good example for me but I am looking for further
> ones. Of course, we also use super-resolution techniques in our facility
> but my observation is that these are used rather to provide "one nice image
> for a publication" or "another piece in the puzzle of evidences" but they
> are not "game winners", they were not necessarily the major piece of
> evidence to prove a biological hypothesis. So I am looking for biological
> questions that could be answered "only"/mostly by the existing
> super-resolution methods.
>         Thanks a lot for your help!
>
> Greetings                       Gabor
>
>

[Tanneke den Blaauwen]

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Dear Gabor,

The discovery that the FtsZ-ring in bacteria is not a ring and that FtsZ is tread milling… (although the latter was also shown by 3D-SIM)

Buss, J., Coltharp, C., Huang, T., Pohlmeyer, C., Wang, S.-C., Hatem, C., & Xiao, J. (2013). In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy. Molecular Microbiology, 89(6), 1099–1120. http://doi.org/10.1111/mmi.12331
Yang, X., Lyu, Z., Miguel, A., McQuillen, R., Huang, K. C., & Xiao, J. (2017). GTPase activity-coupled treadmilling of the bacterial tubulin FtsZ organizes septal cell wall synthesis. Science (New York, NY), 355(6326), 744–747. http://doi.org/10.1126/science.aak9995 <http://doi.org/10.1126/science.aak9995>


Kind regards,

Tanneke

> On 19 Aug 2017, at 16:46, Csúcs Gábor <[hidden email]> wrote:
>
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> *****
>
>
> Dear All,
>
> I would have a slightly provocative question to the community: Could you help me to identify significant/major biological discoveries that were clearly dependent on the availability of various super-resolution light microscopy methods? The PNAS paper from the Zhuang lab (2016) about the actin-spectrin ring is a good example for me but I am looking for further ones. Of course, we also use super-resolution techniques in our facility but my observation is that these are used rather to provide "one nice image for a publication" or "another piece in the puzzle of evidences" but they are not "game winners", they were not necessarily the major piece of evidence to prove a biological hypothesis. So I am looking for biological questions that could be answered "only"/mostly by the existing super-resolution methods.
> Thanks a lot for your help!
>
> Greetings Gabor
>

-------"Guardare, meravigliarsi e tornar a guardare."-------------


Dr. Tanneke den Blaauwen
Associate prof.
Bacterial Cell Biology
Swammerdam Institute for Life Sciences
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University of Amsterdam
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[Alberto Diaspro]

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well, another example is related to
the mechanisms of inhibitory synaptic plasticity

see: https://www.researchgate.net/publication/312204377_Nanoscale_molecular_reorganization_of_the_inhibitory_postsynaptic_density_is_a_determinant_of_GABAergic_synaptic_potentiation
best
alby

> Il giorno 19 ago 2017, alle ore 16:46, Csúcs Gábor <[hidden email]> ha scritto:
>
> *****
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> *****
>
>
> Dear All,
>
> I would have a slightly provocative question to the community: Could you help me to identify significant/major biological discoveries that were clearly dependent on the availability of various super-resolution light microscopy methods? The PNAS paper from the Zhuang lab (2016) about the actin-spectrin ring is a good example for me but I am looking for further ones. Of course, we also use super-resolution techniques in our facility but my observation is that these are used rather to provide "one nice image for a publication" or "another piece in the puzzle of evidences" but they are not "game winners", they were not necessarily the major piece of evidence to prove a biological hypothesis. So I am looking for biological questions that could be answered "only"/mostly by the existing super-resolution methods.
> Thanks a lot for your help!
>
> Greetings Gabor
>

[0000001ed7f52e4a-dmarc-request]

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Many surface receptors have been found to be clustered on the plasma membrane, while some were known to be clustered from electron microscopy studies, with PALM this was extended to live cells.
see e.g. Lillemeier et al. http://www.nature.com/ni/journal/v11/n1/abs/ni.1832.html?foxtrotcallback=true

We have shown on fixed cells that the B cell receptor, CD19 and CD22 are found in nanometer sized clusters.
https://www.researchgate.net/publication/236053888_The_Actin_and_Tetraspanin_Networks_Organize_Receptor_Nanoclusters_to_Regulate_B_Cell_Receptor-Mediated_Signaling
https://www.researchgate.net/publication/286924448_Nanoscale_organization_and_dynamics_of_the_siglec_CD22_cooperate_with_the_cytoskeleton_in_restraining_BCR_signalling

Well, I think there will need more to be done in this area and only time will tell if these then count as significant/major biological discoveries...

best wishes

Andreas




-----Original Message-----
From: Csúcs Gábor <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Sat, 19 Aug 2017 15:53
Subject: Usefulness of super-resolution?

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Dear All,

I would have a slightly provocative question to the community: Could you help me to identify significant/major biological discoveries that were clearly dependent on the availability of various super-resolution light microscopy methods? The PNAS paper from the Zhuang lab (2016) about the actin-spectrin ring is a good example for me but I am looking for further ones. Of course, we also use super-resolution techniques in our facility but my observation is that these are used rather to provide "one nice image for a publication" or "another piece in the puzzle of evidences" but they are not "game winners", they were not necessarily the major piece of evidence to prove a biological hypothesis. So I am looking for biological questions that could be answered "only"/mostly by the existing super-resolution methods.
        Thanks a lot for your help!

Greetings Gabor

[Ewers, Helge]

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Hey Gabor,

there is the organisation of the y-complex within the nuclear pore (Szymborska, Science, 2013), a very important problem. The growth of dendritic spines in response to signalling, which was completely handwaving before (Tonnensen, Nature Neuroscience, 2014), and of course the amazing works on focal adhesion complexes by Waterman-Storer, Hess and Kanchanawong (Nature, 2010, Nature Cell Biology, 2015, Nature Cell Biology 2017). And the decade worth of Synaptic receptor organization by the Choquet lab. And distribution of integrins by the Giannone lab.
The groundbreaking quantitative analysis of organelle contact sites by Jennifer Lippincott Schwartz, Betzig et al, (Calm et al, Nature, 2017), the suborganization of clathrin coated pit components by Hess and Taraska (Sochacki et al, Nature Cell Biology 2017).
All these were inaccessible problems before. Although less prominent, I think it is pretty cool that we could show how individual septin complexes line up within filaments in cells (Kaplan et al., Nano Letters 2015).

Just to name a few striking and prominent examples. By now, you can find excellent biological important work i.e. in MBC all the time.

Best wishes to Zurich,

I ‘ll be there this weekend, if you want to chat,

Helge



--

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Professor for Membrane Biochemistry
Freie Universität Berlin
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On 19 Aug 2017, at 16:46, Csúcs Gábor <[hidden email]<mailto:[hidden email]>> wrote:

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Dear All,

I would have a slightly provocative question to the community: Could you help me to identify significant/major biological discoveries that were clearly dependent on the availability of various super-resolution light microscopy methods? The PNAS paper from the Zhuang lab (2016) about the actin-spectrin ring is a good example for me but I am looking for further ones. Of course, we also use super-resolution techniques in our facility but my observation is that these are used rather to provide "one nice image for a publication" or "another piece in the puzzle of evidences" but they are not "game winners", they were not necessarily the major piece of evidence to prove a biological hypothesis. So I am looking for biological questions that could be answered "only"/mostly by the existing super-resolution methods.
Thanks a lot for your help!

Greetings Gabor

[Reto Fiolka]

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Dear Gabor and Helge,


I would not consider the quantitative analysis of organelle contact sites by Jennifer Lippincott Schwartz, Betzig et al, (Valm et al, Nature, 2017) as an application of superresolution microscopy. Per methods section, the resolution ranged from 294 microns (lateral) x 649 microns (axial) to 370 microns (lateral) x 947 microns (axial) for the wavelength range of 445-642nm.
Thus confocal-scale axial resolution and a bit poorer lateral resolution. This should by no means discredit the great effort in live multicolor imaging, it just does not fit to the list of superresolution applications.



Best,
Reto

[Ewers, Helge]

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Hi Reto,

good to hear from you. Hope all is good at Southwestern.

Spatial resolution was not “super” strictly indeed, but the combined temporal and spatial resolution in the LLS experiments was only achievable by newly developed microscopy techniques and this is I believe an excellent showcase of what cell biology is going to be due to these newly developed approaches. So I think it is a fair answer in the spirit of Gabors question as to why we go through developing all these complicated new machines and what does the hype deliver.

More to see next week at the SMLMS2017. This time in London organised by the great Dylan Owen and Ricardo Henriques BTW. Hope to see many familiar faces.

Cheers,

Helge




--
 
 
Dr. Helge Ewers
Senior Lecturer
King's College London
Randall Division of Cell and Molecular Biophysics
 2nd Floor, Hodgkin Building
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> On 22 Aug 2017, at 02:53, Reto Fiolka <[hidden email]> wrote:
>
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> *****
>
> Dear Gabor and Helge,
>
>
> I would not consider the quantitative analysis of organelle contact sites by Jennifer Lippincott Schwartz, Betzig et al, (Valm et al, Nature, 2017) as an application of superresolution microscopy. Per methods section, the resolution ranged from 294 microns (lateral) x 649 microns (axial) to 370 microns (lateral) x 947 microns (axial) for the wavelength range of 445-642nm.
> Thus confocal-scale axial resolution and a bit poorer lateral resolution. This should by no means discredit the great effort in live multicolor imaging, it just does not fit to the list of superresolution applications.
>
>
>
> Best,
> Reto
>

[Oliver Biehlmaier-2]

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Dear all,

Thank you for compiling that list.
I think what it shows is that biological discoveries dependent on the availability of super-resolution light microscopy methods were (so far) mainly achieved by groups that are developing super-resolution microscopy and are thus at the forefront of SR since years.

From our facility experience, I confirm Gabor’s observation: Most of the users (>10) that we had at our 3D-SIM system requested only a few assisted imaging slots to acquire this additional “nice image” for publication. So far (in almost 5 years of usage) only 2 groups were really dependent on 3D-SIM in order to answer one of the key questions in  their project (e.g. visualise a specific protein localisation; Roth et al, Nat Commun, 2015, PMID: 25791062).
However, I have the impression that the number of users that are willing to dive into a project that is mainly based on super-resolution microscopy is about to grow. I guess that this number will increase with the simplification of super-resolution microscope usage. Most of the researchers are capable to provide almost perfect samples for super-resolution (following a provided protocol) but only few are willing to learn the physical basics needed to run the experiment on the microscope themselves.

Best,
Oliver



Oliver Biehlmaier, PhD | Head of Imaging Core Facility  | Biozentrum, University of Basel | Klingelbergstrasse 50/70 | CH-4056 Basel
Phone: [phone] | Email: [hidden email]<mailto:[hidden email]> | www.biozentrum.unibas.ch/imcf<http://www.biozentrum.unibas.ch/imcf> | www.microscopynetwork.unibas.ch<http://www.microscopynetwork.unibas.ch>




On 19 Aug 2017, at 16:46, Csúcs Gábor <[hidden email]<mailto:[hidden email]>> wrote:

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Dear All,

I would have a slightly provocative question to the community: Could you help me to identify significant/major biological discoveries that were clearly dependent on the availability of various super-resolution light microscopy methods? The PNAS paper from the Zhuang lab (2016) about the actin-spectrin ring is a good example for me but I am looking for further ones. Of course, we also use super-resolution techniques in our facility but my observation is that these are used rather to provide "one nice image for a publication" or "another piece in the puzzle of evidences" but they are not "game winners", they were not necessarily the major piece of evidence to prove a biological hypothesis. So I am looking for biological questions that could be answered "only"/mostly by the existing super-resolution methods.
Thanks a lot for your help!

Greetings Gabor

[Gwosch, Klaus]

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Dear Gabor,

As you mentioned, the PNAS paper from the Zhuang lab is an excellent example of something that can be seen ONLY with super-resolution.
I would like to point out that that paper is just one of a series of works in which the Zhuang’s and Hell’s lab characterize unprecedented features regarding the organization of the cytoskeleton, adhesion molecules and even channels throughout the nervous system both in (living) cultured cells and in tissues.

http://science.sciencemag.org/content/339/6118/452.full
https://elifesciences.org/articles/04581
http://www.cell.com/cell-reports/abstract/S2211-1247(15)00134-5
https://www.nature.com/articles/srep22741
http://www.pnas.org/content/114/2/E191.short

Other prominent examples of discoveries allowed by super resolution are
- The organization of proteins on synaptic vesicles https://www.nature.com/nature/journal/v440/n7086/full/nature04592.html
- The ultrastructural organization of the neuromuscular synapse in Drosophila http://science.sciencemag.org/content/312/5776/1051.full
- The organization of mitochondrial proteins http://www.pnas.org/content/110/22/8936.abstract and the formation of BAX rings on the mitochondria during the apoptotic process http://emboj.embopress.org/content/early/2016/01/14/embj.201592789
- The structure of transverse tubules (TTs) in cardiomyocytes https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219578/

Best,
Klaus


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Csúcs Gábor
Sent: Saturday, August 19, 2017 4:47 PM
To: [hidden email]
Subject: Usefulness of super-resolution?

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Dear All,

I would have a slightly provocative question to the community: Could you help me to identify significant/major biological discoveries that were clearly dependent on the availability of various super-resolution light microscopy methods? The PNAS paper from the Zhuang lab (2016) about the actin-spectrin ring is a good example for me but I am looking for further ones. Of course, we also use super-resolution techniques in our facility but my observation is that these are used rather to provide "one nice image for a publication" or "another piece in the puzzle of evidences" but they are not "game winners", they were not necessarily the major piece of evidence to prove a biological hypothesis. So I am looking for biological questions that could be answered "only"/mostly by the existing super-resolution methods.
        Thanks a lot for your help!

Greetings                       Gabor

[Kirti Prakash-2]

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Dear all,
Superresolution microscopy has also brought a lot of new insights into chromatin organisation which was missing from the discussion (Bohn et al. 2010, Ricci et al. 2015, Boettiger et al. 2016).
http://www.sciencedirect.com/science/article/pii/S0006349510007319
http://www.sciencedirect.com/science/article/pii/S0092867415001324
https://www.nature.com/nature/journal/v529/n7586/full/nature16496.html

Superresolution further helped to find new structural features of chromatin. We found that during interphase, chromatin dynamically gets remodelled upon stress and adapts to a hollow, condensed ring and rod-like configuration, which reverses back to the initial structure when stress conditions cease (Kirmes et al. 2015). During meiosis, chromatin arranges itself into periodic and distinct functional compartments, indicating the spatial organisation of active and inactive regions of the genome (Prakash et al. 2015). These features were not possible to see with conventional microscopes.
https://genomebiology.biomedcentral.com/articles/10.1186/s13059-015-0802-2
http://www.pnas.org/content/112/47/14635.short

So, definitely super resolution microscopy has brought a lot of novel insights in various areas of biology.

Regarding concerns of Oliver and Gabor, I think most super resolution microscopes require a lot of technical knowledge, and their expensive cost makes a lot of microscopy users think twice before using them. In this regard, I recently showed that a conventional epifluorescence setup could be used to generate high-resolution single molecule images.
http://www.biorxiv.org/content/biorxiv/early/2017/05/17/121061.full.pdf

Another attractive option is from ONI guys, who have come up with a desktop size microscope to do super resolution. I recently tried their microscope, and apart from changing the typical microscope prototype (with oculars, big size, etc.), the desktop microscope is very stable and easy to use (no alignment, etc.). I believe this to be the future of microscopy as the institute and lab space gets smaller and smaller.

I hope such alternative, relatively cheaper solutions can bring super resolution to the masses.  

Best,
Kirti