Fixation artefacts when imaging actin cortex and transmembrane proteins by Super-Resolution

[Ricardo Henriques]

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Dear colleagues,

I wanted to do a small highlight on a preprint we've uploaded to bioRxiv yesterday:
"Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation"
https://www.biorxiv.org/content/early/2018/10/23/450635

With Super-Resolution we show careful fixation is needed to correctly represent the actin cortex and membrane receptor topology. Suboptimal fixation easily leads to artefacts. Hope this may be of interest to you.

All the best,
-Ricardo

--
Ricardo Henriques, Associate Professor
University College London and The Francis Crick Institute

[https://docs.google.com/uc?export=download&id=0BzbBSUuAJup_NDRXcWM0T1VpQXc&revid=0BzbBSUuAJup_ZzVnRS9EK3FwTUNFbnVhdmx5Y1Azbjl4NytZPQ]<http://www.ucl.ac.uk/lmcb/research-group/ricardo-henriques-research-group>
MRC-Laboratory for Molecular Cell Biology
University College London
Gower Street
London WC1E 6BT
United Kingdom

Twitter: @HenriquesLab<https://twitter.com/HenriquesLab>
ORCID: https://orcid.org/0000-0002-2043-5234<https://orcid.org/0000-0002-2043-5234/print>
Google Scholar: https://scholar.google.co.uk/citations?user=-peQ4ZsAAAAJ&hl=en

[Cammer, Michael]

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Perhaps of interest:  http://microscopynotes.com/IRM/index.html 

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Henriques, Ricardo
Sent: Wednesday, October 24, 2018 1:57 PM
To: [hidden email]
Subject: Fixation artefacts when imaging actin cortex and transmembrane proteins by Super-Resolution


Dear colleagues,

I wanted to do a small highlight on a preprint we've uploaded to bioRxiv yesterday:

"Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation"

https://urldefense.proofpoint.com/v2/url?u=https-3A__www.biorxiv.org_content_early_2018_10_23_450635&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=mWNqUGoeqFzAXCeVNVCIuV-VgJfkt_oi5Oj-qjTX-HQ&e=



With Super-Resolution we show careful fixation is needed to correctly represent the actin cortex and membrane receptor topology. Suboptimal fixation easily leads to artefacts. Hope this may be of interest to you.



All the best,

-Ricardo



--

Ricardo Henriques, Associate Professor

University College London and The Francis Crick Institute



[https://urldefense.proofpoint.com/v2/url?u=https-3A__docs.google.com_uc-3Fexport-3Ddownload-26id-3D0BzbBSUuAJup-5FNDRXcWM0T1VpQXc-26revid-3D0BzbBSUuAJup-5FZzVnRS9EK3FwTUNFbnVhdmx5Y1Azbjl4NytZPQ&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=2ghNHJsNSekUKC7VvcBxFwUJU6Nx-K3hz88BbmSwmTM&e=]<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ucl.ac.uk_lmcb_research-2Dgroup_ricardo-2Dhenriques-2Dresearch-2Dgroup&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=NIrZnYPglsq5_eN8_PwptRP0o2GRveQ8T7ZjAMhY2bg&e=>

MRC-Laboratory for Molecular Cell Biology

University College London

Gower Street

London WC1E 6BT

United Kingdom



Twitter: @HenriquesLab<https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_HenriquesLab&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=wnVufIovrOOX9vlVRVQVXeteG_NeJ2dlOlKaqrLmFjM&e=>

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[JAMES B PAWLEY]

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*****

Thanks for the reminder Ricardo.

If you aim at electron microscope resolution on fixed specimens, you need to use electron microscope preparative procedures.

This is why we included Chapter 18 in The Handbook.

GUIDING PRINCIPLES OF SPECIMEN PRESERVATION FOR CONFOCAL FLUORESCENCE MICROSCOPY
by Robert Bacallao, Sadaf Sohrab, and Carrie Phillips

Perhaps others may find this chapter useful.

Best,

Jim Pawley

James and Christine Pawley, 5446 Burley Place, Box 2348, Sechelt BC, Canada, V0N3A0 [hidden email]<mailto:[hidden email]>, Phone [phone], cell 1-604-989-6146



On Oct 24, 2018, at 12:33 PM, Cammer, Michael <[hidden email]<mailto:[hidden email]>> wrote:

*****
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*****

Perhaps of interest:  http://microscopynotes.com/IRM/index.html

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Henriques, Ricardo
Sent: Wednesday, October 24, 2018 1:57 PM
To: [hidden email]
Subject: Fixation artefacts when imaging actin cortex and transmembrane proteins by Super-Resolution


Dear colleagues,

I wanted to do a small highlight on a preprint we've uploaded to bioRxiv yesterday:

"Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation"

https://urldefense.proofpoint.com/v2/url?u=https-3A__www.biorxiv.org_content_early_2018_10_23_450635&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=mWNqUGoeqFzAXCeVNVCIuV-VgJfkt_oi5Oj-qjTX-HQ&e=



With Super-Resolution we show careful fixation is needed to correctly represent the actin cortex and membrane receptor topology. Suboptimal fixation easily leads to artefacts. Hope this may be of interest to you.



All the best,

-Ricardo



--

Ricardo Henriques, Associate Professor

University College London and The Francis Crick Institute



[https://urldefense.proofpoint.com/v2/url?u=https-3A__docs.google.com_uc-3Fexport-3Ddownload-26id-3D0BzbBSUuAJup-5FNDRXcWM0T1VpQXc-26revid-3D0BzbBSUuAJup-5FZzVnRS9EK3FwTUNFbnVhdmx5Y1Azbjl4NytZPQ&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=2ghNHJsNSekUKC7VvcBxFwUJU6Nx-K3hz88BbmSwmTM&e=]<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ucl.ac.uk_lmcb_research-2Dgroup_ricardo-2Dhenriques-2Dresearch-2Dgroup&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=NIrZnYPglsq5_eN8_PwptRP0o2GRveQ8T7ZjAMhY2bg&e=>

MRC-Laboratory for Molecular Cell Biology

University College London

Gower Street

London WC1E 6BT

United Kingdom



Twitter: @HenriquesLab<https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_HenriquesLab&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=wnVufIovrOOX9vlVRVQVXeteG_NeJ2dlOlKaqrLmFjM&e=>

ORCID: https://urldefense.proofpoint.com/v2/url?u=https-3A__orcid.org_0000-2D0002-2D2043-2D5234&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=W-j-Zbz3To3v38rmRWcc6wgZ_8YeWuw05tdB6zVZKm4&e=<https://urldefense.proofpoint.com/v2/url?u=https-3A__orcid.org_0000-2D0002-2D2043-2D5234_print&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=5c_uGFkk1I9y2LuS1cHBWwUjH7eAk9sXysg4lCX2l-U&e=>

Google Scholar: https://urldefense.proofpoint.com/v2/url?u=https-3A__scholar.google.co.uk_citations-3Fuser-3D-2DpeQ4ZsAAAAJ-26hl-3Den&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=VOmHzLKr3Xv3NJmre7fEUUjxqmAnAOOXw7FTg6C0hmU&e=


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[George McNamara]

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*****

Speaking of IRM and general reflection, see

https://www.rms.org.uk/study-read/infocus-magazine/infocus-listing/reflection-contrast-microscopy-review.html


          1 September 2017Issue 47

PLOEM J.S. and PRINS F.A.


    Reflection-Contrast Microscopy - Review

This article gives an overview of the applications of
Reflection-Contrast Microscopy (RCM).

DOI: 10.22443/rms.inf.1.152

A short explanation of the antiflex and oblique illumination methods,
used with RCM is presented. The advantage of using ultrathin sections
for light microscopy with RCM is discussed. Further details can also be
found on the website: www.ploem-reflection-contrast-microscopy.com.

A video illustrating RCM is available on YouTube:
www.youtube.com/watch?v=sNQGu3nDPX8

***
All RMS InFocus articles are open access after 1 year. Same issue has
light sheet review and an earlier issue has Raspberry Pi.

enjoy,
George
p.s. confocal IRM HeLa https://works.bepress.com/gmcnamara/10/

On 10/24/2018 3:33 PM, Cammer, Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Perhaps of interest:  http://microscopynotes.com/IRM/index.html
>
> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]> On Behalf Of Henriques, Ricardo
> Sent: Wednesday, October 24, 2018 1:57 PM
> To: [hidden email]
> Subject: Fixation artefacts when imaging actin cortex and transmembrane proteins by Super-Resolution
>
>
> Dear colleagues,
>
> I wanted to do a small highlight on a preprint we've uploaded to bioRxiv yesterday:
>
> "Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation"
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.biorxiv.org_content_early_2018_10_23_450635&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=mWNqUGoeqFzAXCeVNVCIuV-VgJfkt_oi5Oj-qjTX-HQ&e=
>
>
>
> With Super-Resolution we show careful fixation is needed to correctly represent the actin cortex and membrane receptor topology. Suboptimal fixation easily leads to artefacts. Hope this may be of interest to you.
>
>
>
> All the best,
>
> -Ricardo
>
>
>
> --
>
> Ricardo Henriques, Associate Professor
>
> University College London and The Francis Crick Institute
>
>
>
> [https://urldefense.proofpoint.com/v2/url?u=https-3A__docs.google.com_uc-3Fexport-3Ddownload-26id-3D0BzbBSUuAJup-5FNDRXcWM0T1VpQXc-26revid-3D0BzbBSUuAJup-5FZzVnRS9EK3FwTUNFbnVhdmx5Y1Azbjl4NytZPQ&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=2ghNHJsNSekUKC7VvcBxFwUJU6Nx-K3hz88BbmSwmTM&e=]<https://urldefense.proofpoint.com/v2/url?u=http-3A__www.ucl.ac.uk_lmcb_research-2Dgroup_ricardo-2Dhenriques-2Dresearch-2Dgroup&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=NIrZnYPglsq5_eN8_PwptRP0o2GRveQ8T7ZjAMhY2bg&e=>
>
> MRC-Laboratory for Molecular Cell Biology
>
> University College London
>
> Gower Street
>
> London WC1E 6BT
>
> United Kingdom
>
>
>
> Twitter: @HenriquesLab<https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_HenriquesLab&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=wnVufIovrOOX9vlVRVQVXeteG_NeJ2dlOlKaqrLmFjM&e=>
>
> ORCID: https://urldefense.proofpoint.com/v2/url?u=https-3A__orcid.org_0000-2D0002-2D2043-2D5234&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=W-j-Zbz3To3v38rmRWcc6wgZ_8YeWuw05tdB6zVZKm4&e=<https://urldefense.proofpoint.com/v2/url?u=https-3A__orcid.org_0000-2D0002-2D2043-2D5234_print&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=5c_uGFkk1I9y2LuS1cHBWwUjH7eAk9sXysg4lCX2l-U&e=>
>
> Google Scholar: https://urldefense.proofpoint.com/v2/url?u=https-3A__scholar.google.co.uk_citations-3Fuser-3D-2DpeQ4ZsAAAAJ-26hl-3Den&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=VOmHzLKr3Xv3NJmre7fEUUjxqmAnAOOXw7FTg6C0hmU&e=
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================
>

[Ingela Parmryd-3]

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Dear all,

The importance of fixation for the preservation of molecular organisation is an important topic so it is good that it gets revisited. For the actin cytoskeleton it was acknowledged long before the introduction of superresolution microscopy that the care must be taken and great effort was invested in the development of filament-preserving fixation protocols (see for instance Small Cytoskeleton buffer<https://www.ncbi.nlm.nih.gov/pubmed/6799521>). In the field it is well-established that the use of 4% PFA is not appropriate.
Another problem with fixation is that a change in temperature can cause the redistribution of cellular components so unless fixation is performed at physiological temperature, the observed distributions may not reflect the in vivo scenario. This we have shown for both cholesterol and proteins involved in T cell signalling (Cholesterol<https://www.ncbi.nlm.nih.gov/pubmed/18373974>; T cell signalling proteins<https://www.ncbi.nlm.nih.gov/pubmed/16014381>).
Kind regards,

Ingela Parmryd

> -----Original Message-----
> From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> On Behalf Of Henriques, Ricardo
> Sent: Wednesday, October 24, 2018 1:57 PM
> To: [hidden email]<mailto:[hidden email]>
> Subject: Fixation artefacts when imaging actin cortex and transmembrane proteins by Super-Resolution
>
>
> Dear colleagues,
>
> I wanted to do a small highlight on a preprint we've uploaded to bioRxiv yesterday:
>
> "Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation"
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__www.biorxiv.org_content_early_2018_10_23_450635&d=DwIGaQ&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=oU_05LztNstAydlbm5L5GDu_vAdjXk3frDLx_CqKkuo&m=rR_TAsuHCQvOR7r44A0KnTFHHb4TJ2AwzNGzpKasbzg&s=mWNqUGoeqFzAXCeVNVCIuV-VgJfkt_oi5Oj-qjTX-HQ&e=
>
>
>
> With Super-Resolution we show careful fixation is needed to correctly represent the actin cortex and membrane receptor topology. Suboptimal fixation easily leads to artefacts. Hope this may be of interest to you.
>
>
>
> All the best,
>
> -Ricardo
>
>
>
> --
>
> Ricardo Henriques, Associate Professor
>
> University College London and The Francis Crick Institute
>
>
>
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