nuclear marking fluorophores - for simpler image analysis

[Jeremy Adler-4]

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Dapi and similar fluorophores are regularly used to highlight nuclei.
But the staining is inhomogenous within nuclei and can vary substantially between nuclei in the same specimen, which makes segmentation for quantitative analysis difficult.
So any suggestions for a marker that produces a more uniform fluorescence within individual nuclei and less variation between nuclei, which would make image analysis simpler when we only want to know where the nuclei are.

Jeremy Adler
BioVis
Uppsala U








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[George McNamara]

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Hi Jeremy,

Propidium iodide (PI) and To-Pro-3 have few 'details'. Broad emission
spectra, should be spectrally (and FLIM) unmixable. I encourage our
users here to use Zombie NIR (BioLegend) both because way out in near
infrared and cool name. PicoGreen is very interesting because can be
used at low concentration and 'light up' mitochondrial nucleoids (in
addition to nuclei) in live cells. Uno 2021 Nature Communications
article 2650 (https://www.nature.com/articles/s41467-021-23019-w)
published a new dye that they like over PicoGreen (synthesized by their
lab).

anti-Histone (or antibody to mCherry est for FP-histone H2B) also possible.

Segmentation: no DNA counterstain is going to solve the problem of close
together nuclei. I suggest ditching DNA counterstain and moving to
nuclear pore complex antigens or nuclear lamins. NPCs also provide
discrete structures that either work as point spread function targets or
as resolution (or precision localization) test targets.


happy 2021,

George

p.s. multiplex ... no need to be stuck on DAPI, green, red, NIR ... for
example, Germain lab 2020 PNAS published 65plex (up to 11plex per
iteration) https://www.pnas.org/content/117/52/33455.long

more suggestions at http://confocal.jhu.edu/mctips/multiplex



On 6/2/2021 6:31 AM, Jeremy Adler 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.
> *****
>
> Dapi and similar fluorophores are regularly used to highlight nuclei.
> But the staining is inhomogenous within nuclei and can vary substantially between nuclei in the same specimen, which makes segmentation for quantitative analysis difficult.
> So any suggestions for a marker that produces a more uniform fluorescence within individual nuclei and less variation between nuclei, which would make image analysis simpler when we only want to know where the nuclei are.
>
> Jeremy Adler
> BioVis
> Uppsala U
>
>
>
>
>
>
>
>
> När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/
>
> E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/en/about-uu/data-protection-policy

[Abby Dernburg]

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I second the recommendation to use a nuclear periphery stain for segmentation. If you’re working with metazoan cells, lamin antibodies give a smoother signal than nuclear pores but either is useful.

Abby Dernburg, Ph.D.
HHMI Investigator
Department of Molecular and Cell Biology
University of California, Berkeley
Berkeley, CA 94720
USA

Mobile: [phone]

> On Jun 2, 2021, at 4:35 AM, George McNamara <[hidden email]> 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.
> *****
>
> Hi Jeremy,
>
> Propidium iodide (PI) and To-Pro-3 have few 'details'. Broad emission spectra, should be spectrally (and FLIM) unmixable. I encourage our users here to use Zombie NIR (BioLegend) both because way out in near infrared and cool name. PicoGreen is very interesting because can be used at low concentration and 'light up' mitochondrial nucleoids (in addition to nuclei) in live cells. Uno 2021 Nature Communications article 2650 (https://www.nature.com/articles/s41467-021-23019-w) published a new dye that they like over PicoGreen (synthesized by their lab).
>
> anti-Histone (or antibody to mCherry est for FP-histone H2B) also possible.
>
> Segmentation: no DNA counterstain is going to solve the problem of close together nuclei. I suggest ditching DNA counterstain and moving to nuclear pore complex antigens or nuclear lamins. NPCs also provide discrete structures that either work as point spread function targets or as resolution (or precision localization) test targets.
>
>
> happy 2021,
>
> George
>
> p.s. multiplex ... no need to be stuck on DAPI, green, red, NIR ... for example, Germain lab 2020 PNAS published 65plex (up to 11plex per iteration) https://www.pnas.org/content/117/52/33455.long
>
> more suggestions at http://confocal.jhu.edu/mctips/multiplex
>
>
>
> On 6/2/2021 6:31 AM, Jeremy Adler 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.
>> *****
>>
>> Dapi and similar fluorophores are regularly used to highlight nuclei.
>> But the staining is inhomogenous within nuclei and can vary substantially between nuclei in the same specimen, which makes segmentation for quantitative analysis difficult.
>> So any suggestions for a marker that produces a more uniform fluorescence within individual nuclei and less variation between nuclei, which would make image analysis simpler when we only want to know where the nuclei are.
>>
>> Jeremy Adler
>> BioVis
>> Uppsala U
>>
>>
>>
>>
>>
>>
>>
>>
>> När du har kontakt med oss på Uppsala universitet med e-post så innebär det att vi behandlar dina personuppgifter. För att läsa mer om hur vi gör det kan du läsa här: http://www.uu.se/om-uu/dataskydd-personuppgifter/
>>
>> E-mailing Uppsala University means that we will process your personal data. For more information on how this is performed, please read here: http://www.uu.se/en/about-uu/data-protection-policy

[Sylvie Le Guyader]

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Hi Jeremy

We like to use laminA antibodies. Robust staining and easy to segment. :)

Med vänlig hälsning / Best regards
Sylvie
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Blickagången 16,
Room 7362 (lab)/7840 (office)
14157 Huddinge, Sweden
mobile: [phone]
LCI website
LCI microscopy blog

-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jeremy Adler
Sent: Wednesday, 2 June, 2021 12:32
To: [hidden email]
Subject: nuclear marking fluorophores - for simpler image analysis

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Dapi and similar fluorophores are regularly used to highlight nuclei.
But the staining is inhomogenous within nuclei and can vary substantially between nuclei in the same specimen, which makes segmentation for quantitative analysis difficult.
So any suggestions for a marker that produces a more uniform fluorescence within individual nuclei and less variation between nuclei, which would make image analysis simpler when we only want to know where the nuclei are.

Jeremy Adler
BioVis
Uppsala U








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